Tiago B uno Fe ei a Adeno i us as a Biopha maceu ical: Op imisa ion o ec o p oduc ion
Adeno i us as a Biopha maceu ical
Disse a ion p esen ed o ob ain a Ph.D deg ee in Chemical Enginee ing a he Ins i u o de
Tecnologia Química e Biológica, Uni e sidade No a de Lisboa
Tiago B uno Fe ei a
Op imisa ion o ec o p oduc ion
Oei as, Ma ch 2007
Vaccina ion has been one o he mos impo an
in e en ions designed o p e en disease o be
employed on a wo ldwide basis. Adeno i al ec o
has been ex ensi ely s udied as a accine pla o m
because o i s abili y o induce po en cellula and
humo al immuni y. Consequen ly, he ma ke
equi emen s o adeno i al ec o s a e inc easing,
c ea ing a need o new me hodologies o la ge-
scale p oduc ion o concen a ed ec o s wi h
wa an ed pu i y and e icacy. This wo k
con ibu es o he imp o emen o he s a e o he
a o adeno i al ec o s p oduc ion p ocess.
Adeno i us as a Biopha maceu ical
Op imisa ion o ec o p oduc ion
By
Tiago B uno Pe ei a Soa es Fe ei a
Disse a ion p esen ed o ob ain a Ph.D
deg ee in Chemical Enginee ing a he
Ins i u o de Tecnologia Química e Biológica,
Uni e sidade No a de Lisboa
Oei as, Ma ch 2007
Adeno i us as a Biopha maceu ical
Op imisa ion o ec o p oduc ion
Tiago B uno Pe ei a Soa es Fe ei a
Disse a ion p esen ed o ob ain a Ph.D deg ee in Chemical
Enginee ing a he Ins i u o de Tecnologia Química e
Biológica, Uni e sidade No a de Lisboa
Supe iso s: Paula M. Al es and Manuel J. T. Ca ondo
Ins i u o de Tecnologia Química e Biológica
Uni e sidade No a de Lisboa
Oei as, Ma ch 2007
ii
Adeno i us as a Biopha maceu ical: Op imisa ion o ec o p oduc ion
Second edi ion: May 2007
Copy igh numbe :
Co e images:
F on : Adeno i us pa icle. Sou ce: image was adap ed om he web si e
h p://nobelp ize.o g/nobel_p izes/medicine/lau ea es/1993/illp es/gene
s-in-pieces.h ml.
Back: Elec on mic oscope pic u e o adeno i us pa icles (up image) and
mic oscope pic u e o 293 cells in ec ed wi h adeno i us ec o s (back
image).
ITQB/IBET Animal Cell Technology Labo a o y
Ins i u o de Tecnologia Química e Biológica/
Ins i u o de Biologia Expe imen al e Tecnológica
Apa ado 12, 2781-901 Oei as, Po ugal
Fax: +351 21 442 11 61; Phone: +351 21 446 94 24
h p://www.i qb.unl.p
h p//www.ibe .p
iii
Supe iso s:
D . Paula Ma ques Al es, P incipal Resea che and G oup Leade o he
Animal Cell Technology Labo a o y a ITQB, Oei as Po ugal.
P o . D . Manuel J. T. Ca ondo, P o esso o Chemical and Biochemical
Enginee ing a Uni e sidade No a de Lisboa, Mon e da Capa ica, Po ugal,
CEO (Chie Execu i e O ice ) o IBET, Oei as, Po ugal, CSO (Chie
Scien i ic O ice ) o GenIBET Biopha maceu icals, Oei as, Po ugal and
Head o he Animal Cell Technology g oup a ITQB, Oei as, Po ugal.
Ju y:
D . John G. Aunins, isi ing P o esso a ITQB, Oei as, Po ugal and Senio
Di ec o o Cell Cul u e De elopmen and Vaccine BioP ocess a Me ck
Resea ch Labs., Philadelphia, USA.
D . E ic J. K eme , G oup Leade and Di ec o o Resea ch a Ins i u de
Géné ique Moléculai e de Mon pellie CNRS, Mon pellie , F ance.
D . Joaquim Sampaio Cab al, P o esso a Ins i u o Supe io Técnico,
Lisboa, Po ugal.
D . Ana So ia Co oadinha, Auxilia Rese che o he associa ed labo a o y
ITQB/IGC/IBET, Oei as, Po ugal.

i
Fo ewo d
The p esen hesis disse a ion is he esul o ou yea s o esea ch a he
Labo a o y o Animal Cell Technology a ITQB/IBET (Po ugal) unde D .
Paula M. Al es and P o . D . Manuel J. T. Ca ondo Supe ision.
This hesis in ends o explo e he limi ing ac o on adeno i us ec o
p oduc ion a high cell densi ies aiming he unde s and and imp o emen
o he cu en ine icien p ocess o adeno i us ec o p oduc ion in o de
o espond o he inc easing demand o me hodologies o he p oduc ion
in la ge scale o concen a ed ec o s wi h wa an ed pu i y and e icacy.
Acknowledgemen s
I would like o exp ess my g a i ude o all he people who di ec ly o
indi ec ly suppo ed and con ibu ed o make possible he wo k p esen ed
in his hesis.
A e y special hanks o my supe iso D . Paula Ma ques Al es, who uly
made a di e ence in my li e. I was unde he u elage ha I de eloped a
ocus and became in e es ed in he animal bio echnology ield. She
p o ided me wi h di ec ion, sugges ions and suppo . I was h ough he
pe sis ence, unde s anding and kindness ha I comple ed his wo k. I
doub ha I will e e be able o con ey my app ecia ion ully. Thank you
e y much.
I would like o exp ess my g a i ude o my supe iso , P o . Manuel
Ca ondo, whose expe ise, unde s anding and pa ience added conside ably
o my g adua e expe ience; o us ing and gi ing me he oppo uni y o
wo k in he animal bio echnology ield. I hank him o he guidance,
suppo , pa ien , igo , p agma ism, mo i a ion and o “pulling he muscle
o i s maximum”. I hank you o his g ea jou ney ha allowed me o
g ow as a scien is and as a pe son.
To D . Ped o E. C uz o all he suppo , mo i a ion, discussions and all
ha he augh me.
I am also ex emely g a e ul o D . John Aunins and D . O o-Wilhelm
Me en o hei inpu .
To my “o ice” lab colleagues Ana So ia Co oadinha and Isabel Ma celino
o hei daily con e sa ions and all he ins ances in which hei iendship
and good mood helped me along he way.
i
To Ana Lúcia Fe ei a and Rica do Pe digão, he ( o me ) diploma s uden s
who g ea ly con ibu ed o he wo k p esen ed in his hesis, o hei
commi men , e o and eamwo k.
I would like o hank a ew people speci ically. An ónio Roldão and Ma cos
Sousa o all he good momen s inside and ou side he lab. To Ca ina
Fe ei a Sil a, always eady o help and o being pa o he “Fe ei a
eam”. To C is ina Peixo o o e e y hing she augh me abou adeno i us
pu i ica ion, o he suppo and iendship. To Change Zhang o all he
suppo . Ma lene Ca mo o he pa ience and o all he help conce ning
he low cy ome ic analyzes. To Rosá io Clemen e o he echnical
Suppo .
I since ely hank all o he cu en and o me membe s o he Animal Cell
Technology Lab o hei help and iendship o e he las ou yea s. I
eally spen a g ea deal o ime a lab.
I would also like o hank my pa en s and b o he o he suppo hey
p o ided me h ough my en i e li e wi hou whose lo e and encou agemen
I would no ha e inished his hesis.
I mus gi e immense hanks o my pa ien and lo ing wi e Alexand a and
ou daugh e Ca olina, who ha e been a g ea sou ce o s eng h all
h ough his wo k, especially du ing hose imes when he esea ch came
i s . Thei lo e and suppo was o immeasu able alue o me.
To he en i ies ha unded he esea ch: Eu opean Commission (P ojec
RP/PPR ORALVAC ICA4-CT-2000-30027), Fundo Social Eu opeu (FSE) and
Fundação pa a a Ciência e a Tecnologia (P ojec POCTI/BIO/46515/2002,
POCI 2010 and s uden g an SFRH/BD/10614/2002).
ii
Abs ac
Adeno i us ec o s (AdV) ha e a ac ed conside able in e es o e he
pas decade, wi h ongoing clinical de elopmen p og ams o applica ions
anging om eplacemen he apy o p o ein de iciencies o cance
he apeu ics o p ophylac ic accines. In ac , in he pe iod 1989-2005,
o e 300 clinical ials we e conduc ed using adeno i uses wo ldwide,
adeno i us ype 5 (Ad5) being he ec o o choice. Consequen ly,
conside able p oduc p ocess, analy ical and o mula ion de elopmen has
o be ca ied ou in o de o espond o he cons an ly inc easing ma ke
equi emen s o AdV.
One o he majo es ic ions on AdV p oduc ion is he so-called “cell
densi y e ec ”, i.e., a d op in cell speci ic p oduc i i y concomi an wi h
inc eased cell concen a ion a in ec ion (CCI) abo e 1106 cells/ml. This
limi a ion on AdV p oduc ion has o be ine- uned in o de o ob ain highe
AdV olume ic p oduc i i ies; his will be he subjec o s udy and
de elopmen in his hesis.
In Chap e I, a s a e o he a on ecen de elopmen s and app oaches o
AdV p oduc ion and pu i ica ion, including successes and ailu es in
e e ina y clinical applica ions o da e, is p esen ed.
In Chap e II, he cell densi y e ec is explo ed. Thus, AdV p oduc ion a
cell densi ies highe han 1×106 cell/ml is e alua ed by compa ing wo
di e en se um ee media (CD 293 medium om In i ogen and EX-Cell
medium om JRH). The esul s show ha while EX-Cell medium allows he
highe maximum cell numbe (7.5×106 cell/ml), CD 293 is he bes op ion
o AdV p oduc ion a high cell densi ies, allowing in ec ion a CCI o 2×106
cell/ml wi hou signi ican losses in e ms o cell speci ic p oduc i i y.
xi
com compos os químicos e in ec adas à mesma CCI. A maio
p oduc i idade celula especí ica oi ob ida quando as células o am
sinc onizadas na ase S do ciclo celula , o que con i ma a nossa hipó ese
an e io . Assim, uma ez que o es ado isiológico das células é um
pa âme o impo an e a conside a na al u a da in ecção na p odução de
AdV, oi desen ol ida uma es a égia pa a sinc oniza células, sem eco e
ao uso de inibido es químicos, usando al e ações da empe a u a. Com es a
es a égia oi possí el ob e uma sinc onização de 57% das células na ase S
do ciclo celula apenas descendo a empe a u a pa a 31ºC du an e 67h e
aumen ando-a no amen e pa a 37ºC du an e 72h. Ao in ec a es as células
sinc onizadas ob e e-se um aumen o de 7.3 ezes na p oduc i idade
celula especí ica. Mas, de ido a pe das na iabilidade celula e aos
ele ados empos de incubação necessá ios pa a ob e sinc onização na ase
S, são necessá ios mais es udos pa a “a ina ” o p ocesso.
Em anexo são mos ados dois es udos adicionais. No Anexo A, é a aliada a
impo ância de ciné icas de in ecção na op imisação do p ocesso de
p odução de AdV, ais como a MOI (Mul iplicidade de In ecção) e HPI (Ho as
Pós In ecção). Em adição, o uso de meios suplemen ados com so o oi
compa ado com um meio li e de so o em e mos de c escimen o celula e
p odução de AdV; no Anexo B, é a aliado o e ei o da adição de i aminas
ou lipidos na al u a da in ecção a ele adas densidades celula es, sem se e
e i icado um aumen o ele an e em e mos de p odução de AdV.
Es a ese ap esen a con ibuições ele an es pa a a ac ual ecnologia de
p odução de AdV. Tan o a limi ação do meio (que po al a de nu ien es
que po p odução excessi a de biop oduc os, nomeadamen e a amónia) e
o es ado isiologico das células no momen o da in ecção o am
iden i icados como pa âme os aliosos a se conside ados na p odução de
AdV a ele adas densidades celula es.

x
Thesis Publica ions
Jou nals
Fe ei a T.B., Al es P.M., Aunins J.G., Ca ondo M.J.T. (2005) “Use o
adeno i al ec o s as e e ina y accines”, Gene The apy, 12:S73-S83.
Fe ei a T.B., Fe ei a A.L., Ca ondo M.J.T., Al es P.M. (2005) “Two
di e en se um- ee media and osmolali y e ec upon human 293 cell
g ow h and adeno i us p oduc ion”, Bio echnology Le e s, 27:1809-
1813.
Fe ei a T.B., Fe ei a A.L., Ca ondo M.J.T., Al es P.M. (2005) “E ec
o e eed s a egies and non-ammoniagenic medium on adeno i us
p oduc ion a high cell densi ies”, Jou nal o Bio echnology, 119:272-
280.
Fe ei a T.B., Ca ondo M.J.T., Al es P.M. (2007) “E ec o ammonia
p oduc ion on in acellula pH: consequen e ec on adeno i us ec o
p oduc ion”, Jou nal o Bio echnology, 129:433-438.
Zhang C., Fe ei a T.B., C uz P.E., Al es P.M., Hau y M., Ca ondo
M.J.T. (2006) “The impo ance o 293 cell cycle phase on adeno i us
ec o p oduc ion”, Enzyme and Mic obial Technology, 39:1328-1332.
Fe ei a T.B., Pe digão R., Sil a A.C., Zhang C., Aunins J.G., Ca ondo
M.J.T., Al es P.M., “293 cell cycle synch onisa ion in adeno i us ec o
p oduc ion”, Bio echnology and Bioenginee ing (submi ed).
Ex ended Con e ence P oceedings
Fe ei a, T.B., Al es, P.M., Gonçal es, D., Ca ondo, M.J.T. (2005)
“E ec o MOI and medium composi ion on adeno i us in ec ion
x i
kine ics”, Animal Cell Technology Mee s Genomics, P oceedings o he
18 h ESACT Mee ing, G anada, Spain, May 11-14, 2003, Edi ed by
Gòdia, F. and Fussenegge , M., Sp inge , 329-332.
Fe ei a T.B., Al es P.M., Ca ondo M.J.T. (2007) “E ec o i amins
o lipids addi ion on adeno i us p oduc ion a high cell densi ies”, in
Smi h R (Ed), Cell Technology P ocesses o Cell P oduc s, Sp inge (in
p ess).
O he Publica ions Desc ibing Wo k o Which he Au ho has
Con ibu ed Du ing his Thesis Wo k bu no Included in his Thesis
Peixo o C., Fe ei a T.B., Ca ondo M.J.T., C uz P.E., Al es P.M.
(2006) “Pu i ica ion o adeno i al ec o s using expanded bed
ch oma og aphy”, Jou nal o Vi ological Me hods, 132:121-126.
Ma ias A., Se a A.T., Malpique R., Sil a A.C., Ma celino I., Fe ei a
T.B., Pe digão R., Al es P., Dua e C., “An i i al E ec o Res e a ol
and a G ape Na u al Ex ac on Adeno i us Type 5 In ec ion”, Jou nal
o Ag icul u al and Food Chemis y (submi ed).
Peixo o C., Fe ei a T.B., Ca ondo M.J.T., Al es P.M., “New
pu i ica ion pla o m o adeno i us ec o s based on memb ane
echnology”, Bio echnology and Applied Biochemis y (submi ed).
x ii
Con en s
CHAPTER I - INTRODUCTION__________________________________ 1
Use o adeno i al ec o s as e e ina y accines________________ 3
CHAPTER II - CELL DENSITY EFFECT___________________________ 43
Two di e en se um- ee media and osmolali y e ec upon human
293 cell g ow h and adeno i us p oduc ion___________________ 45
CHAPTER III - MEDIUM LIMITATIOS ____________________________ 59
Pa 1 - E ec o e eed s a egies and non-ammoniagenic medium
on adeno i us p oduc ion a high cell densi ies________________ 61
Pa 2 - E ec o ammonia p oduc ion on in acellula pH: consequen
e ec on adeno i us ec o p oduc ion______________________ 81
CHAPTER IV - CELL CYCLE _________________________________ 103
Pa 1 - The impo ance o 293 cell cycle phase on adeno i us ec o
p oduc ion ___________________________________________ 105
Pa 2 - 293 cell cycle synch onisa ion in adeno i us ec o
p oduc ion ___________________________________________ 125
CHAPTER V - DISCUSSION AND CONCLUSIONS___________________ 151
CHAPTER VI - ANNEXES____________________________________ 161
Annex A - E ec o MOI and medium composi ion on adeno i us
in ec ion kine ics ______________________________________ 163
Annex B - E ec o i amins o lipids addi ion on adeno i us
p oduc ion a high cell densi ies __________________________ 173
CHAPTER I
In oduc ion

In oduc ion
3
Use o adeno i al ec o s as e e ina y accines
Fe ei a T.B., Al es P.M., Aunins J.G., Ca ondo M.J.T. (2005) Gene The .,
12:S73-S83.
Chap e I
4
Abs ac
Vaccines a e he mos e ec i e and inexpensi e p ophylac ic ool in human
and e e ina y medicine. Ideally, accines should induce a li elong
p o ec i e immuni y agains he a ge pa hogen while no causing clinical
o pa hological signs o diseases in accina ed people o animals. Howe e
such ideal accines a e a e in he e e ina y ield. Many accines a e
ei he o limi ed e ec i eness o ha e ha m ul side e ec s. In addi ion,
he e a e s ill se e e diseases wi h no e ec i e accines. A e y impo an
c i e ion o an ideal accine in e e ina y medicine is low cos ; his is
especially impo an in de eloping coun ies and e en mo e so o poul y
accina ion, whe e accines mus sell o a ew cen s a dose.
T adi ional app oaches include inac i a ed accines, a enua ed li e
accines and subuni accines. Recen ly, gene ic enginee ing has been
applied o design new, imp o ed accines. Adeno i us ec o s a e highly
e icien o gene ans e in a b oad spec um o cell ypes and species.
Mo eo e , adeno i uses o en induce humo al, mucosal and cellula
immune esponses o an igens encoded by he inse ed o eign genes.
Thus, adeno i uses ha e become singula ec o s o choice o deli e y
and exp ession o o eign p o eins o accina ion. Consequen ly, he
ma ke equi emen s o adeno i us accines a e inc easing, c ea ing a
need o p oduc ion me hodologies o concen a ed ec o s wi h
wa an ed pu i y and e icacy. This chap e summa izes ecen
de elopmen s and app oaches o adeno i us p oduc ion and pu i ica ion,
including successes and ailu es in e e ina y clinical applica ions o da e.
A he end o he chap e , a b ie ou line o he scope in ended o his
hesis is p esen ed.
In oduc ion
5
CONTENTS
1. ADENOVIRUS VACCINATION HISTORY _________________________ 6
2. ADENOVIRUS BIOLOGY ____________________________________ 9
3. SPECIES-SPECIFIC PLATFORMS _____________________________ 10
4. APPLICATIONS _________________________________________ 12
4.1. Adeno i us Vaccina ion in companion animals_____________ 12
4.1.1. Feline Immunode iciency Vi us (FIV)_____________________ 12
4.1.2. Canine Dis empe Vi us (CDV) __________________________ 13
4.1.3. Rabies Vi us (RV)_____________________________________ 13
4.2. Adeno i us Vaccina ion in Poul y ______________________ 14
4.2.1. A ian In ec ious B onchi is Vi us (IBV) ___________________ 14
4.2.2. In ec ious Bu sal Disease Vi us (IBDV)____________________ 15
4.3. Adeno i us Vaccina ion in Swine _______________________ 15
4.3.1. Classical Swine Fe e Vi us (CSFV) ______________________ 15
4.3.2. Pseudo abies Vi us (P V)_______________________________ 16
4.3.3. Foo and Mou h Disease Vi us (FMDV)____________________ 17
4.3.4. Po cine Respi a o y and Rep oduc i e Synd ome Vi us (PRRSV)19
4.3.5. T ansmissible Gas oen e i is (Co ona) Vi us (TGEV) _______ 19
4.3.6. Swine In luenza Vi us (SIV)_____________________________ 20
4.4. Adeno i us Vaccina ion in Ca le _______________________ 21
4.4.1. Bo ine Vi al Dia hea Vi us (BVDV) ______________________ 21
4.4.2. Bo ine Pa ain luenza Vi us Type 3 (bPIV3)________________ 22
4.4.3. Bo ine He pes Vi us 1 (BHV-1)__________________________ 23
4.4.4. Rinde pes Vi us (RPV) and Pes e des Pe i s Ruminan s Vi us
(PPRV) ___________________________________________________ 24
5. ADENOVIRUS PRODUCTION AND PURIFICATION AS VETERINARY
VACCINES _______________________________________________ 26
6. SCOPE OF THE THESIS ___________________________________ 30
7. ACKNOWLEDGEMENTS ___________________________________ 31
8. REFERENCES___________________________________________ 32
Chap e I
12
wi h BAd3 ailed o p oduce ei he clinical signs o g oss lesions, bu all
animals se ocon e ed. The cell en y pa hway o BAd3 is no well
unde s ood (Benko and Ha ach 1998; Reddy e al., 2000).
4. APPLICATIONS
4.1. Adeno i us Vaccina ion in companion animals
4.1.1. Feline Immunode iciency Vi us (FIV)
The in ec ion o ca s wi h FIV esul s in an immunosupp essi e disease ha
is ansmi ed by blood and sali a. FIV in ades and des oys he
monocy e/mac ophage sys em and in ec s B-cells (Uhl e al., 2002).
Di e en app oaches ha e been made ying o immunize ca s agains FIV;
so a , no e ec i e accine has been made. Gonin e al., (1995), 10 yea s
ago, cons uc ed a eplica ion-de ec i e HAd5, con aining he en elope
p o ein ENV gene o FIV; howe e , despi e he ac ha an an ibody
esponse o pseudo abies i us in ca s showed he po en ial o HAd5
ec o s o be used in his species, ca s injec ed wi h 1010.8-1011.8 o 50
pe cen issue cul u e in ec ious dose (TCID50) adju an ed wi h mon anide
ISA 708 (wa e in non mine al oil) o wi h mon anide ISA 206 (double
wa e /mine al oil/wa e ) o his AdV did no de elop de ec able an ibody
esponse agains ENV. Mo eo e , i was obse ed ha e en i high i e s o
an ibodies agains ENV p oduc s a e induced, hey could s ill be insu icien
o p o ec ion (Gonin e al., 1995). Since hen, no u he wo k has
appa en ly been pe o med conce ning he use o AdV as a po en ial
accine agains FIV.

In oduc ion
13
4.1.2. Canine Dis empe Vi us (CDV)
CDV induces a al diseases including encephali is wi h demyelina ion,
dia hea and espi a o y diso de s in dogs. Al hough con en ional li e
modi ied accines a e comme cially a ailable and widely used in he ield,
hei e icacy is limi ed in he p esence o ma e nally-de i ed an ibodies
(Ba e 1999). Thus, a new and imp o ed CDV accine which could
o e come his limi a ion would cons i u e a signi ican imp o emen . The
cons uc ion and cha ac e iza ion o he i s eplica ion-compe en CAd2
s a ed ecen ly. The genes ha code o CDV hemagglu inin (HA) (Fische
e al., 2002; Hi ama e al., 2003) o usion (F) (Fische e al., 2002)
p o eins we e inse ed in wo CAd2s and used as a candida e accine in
puppies. I was epo ed ha in anasal accina ion wi h a mix u e
con aining 105.8 TCID50 o which CAd2 p o ided an excellen le el o
p o ec ion in se onega i e puppies, inducing almos comple e p o ec ion.
In con as , in anasal immuniza ion o puppies bo n o CDV and CAd2
immune dams ailed o ac i a e speci ic and p o ec i e immune esponses.
Howe e , when he same puppies we e accina ed subcu aneously,
signi ican se ocon e sion and solid p o ec i e immuni y we e igge ed.
Fu he mo e, a signi ican p iming o memo y esponses was e idenced
immedia ely a e challenge, his cons i u ing an e icien s a egy o
o e come bo h passi e and ac i e Ad speci ic immuni y in he dog.
4.1.3. Rabies Vi us (RV)
Rabies s ill p esen s a heal h h ea no only o humans bu also o dogs
and ca s, being he dog he only impo an ec o o humans, especially in
less de eloped na ions whe e uncon olled canine abies o en is endemic.
A eplica ion-compe en and -de ec i e HAd5 exp essing a abies
glycop o ein (RG) has been de eloped, inducing immuni y o abies in
oden , canine, oxes and skunk when gi en by in amuscula ,
Chap e I
14
subcu aneous o in anasal ou es wi h a dose o 108 TCID50/animal.
Howe e , o al immuniza ion ailed o induce a measu able an ibody
esponse o RV using he same dose (Xiang e al., 1996; Vos e al., 2001).
Dogs p e iously accina ed wi h comme cially a ailable accines,
immunized wi h 107 plaque o ming uni (p u)/animal o a eplica ion-
de ec i e HAd5 exp essing he RG, ha e de eloped highe i e s o i al
neu alizing an ibodies agains RV 10 days a e accina ion when
compa ed wi h con en ional accines unde simila condi ions. Mo eo e ,
he immuniza ion o dogs wi h he comme cial non Ad accines is equi ed
yea ly o , a bes , e e y 3 yea s, depending on he ype o accine. On he
o he hand, he highe an ibody i e s ob ained wi h Ad accines agains RV
would educe he equency o dog immuniza ion, educing he cos s o
pe owne s (Tims e al., 2000). One impo an ad an age o his
ecombinan accine is he ac ha he immune esponse o he RG was
shown no o be impai ed by ma e nal immuni y; hus, his Ad accine is
highly sui able o neona al immuniza ion (Wang e al., 1997).
4.2. Adeno i us Vaccina ion in Poul y
4.2.1. A ian In ec ious B onchi is Vi us (IBV)
IBV is a highly con agious pa hogen o poul y causing signi ican mo bidi y
and mo ali y. Depending on he s ain, IBV can a ge he espi a o y
ac , kidney and o iduc s and esul in neph i is and educed egg
p oduc ion. In addi ion, mo e han 20 IBV se o ypes ha e been iden i ied
wo ldwide and new se o ypic a ian s ha e been iden i ied as a esul o
he widesp ead use o li e a enua ed accines. Di e en app oaches ha e
been de eloped in o de o gene a e a mo e e icacious accine agains
IBV. The exp ession o S1, a glycop o ein in ol ed in he a achmen o
cellula ecep o s, by a accinia i us was able o induce i us-neu alizing
an ibodies o IBV when deli e ed o mice; howe e , mul iple injec ions a e
In oduc ion
15
equi ed o achie e a easonable deg ee o p o ec ion. Recen ly, a FAd
exp essing he S1 o IBV has been de eloped (Johnson e al., 2003). A
single dose o 106 TCID50/animal was shown o be su icien o ob ain
comple e p o ec ion o chickens a he achea, he p ima y si e o
in ec ion by IBV. Mo eo e , e en in he ace o FAV ma e nal an ibodies, a
high le el o p o ec ion was achie ed (Johnson e al., 2003).
4.2.2. In ec ious Bu sal Disease Vi us (IBDV)
IBDV induces an immunosupp essi e disease o chickens by des uc ion o
he B-lymphocy es. Cu en accina ion al e na i es consis o ei he li e
i us o inac i a ed oil-emulsion accines, which induce se um an ibody
p oduc ion in b eeding hens a e na u al exposu e; hey a e ans e ed o
he p ogeny chicks ia he yolk sac p o iding p o ec ion o he i s c i ical
weeks a e ha ching (Mulle e al., 2003). The cons uc ion o a FAd10
con aining he VP2 gene om IBDV has been desc ibed (Sheppa d e al.,
1998). This ecombinan accine was shown o induce an immune esponse
in chickens o VP2 a e accina ion wi h 107 p u/animal. Mo eo e , a e
challenge wi h IBDV, in a enously, in ape i oneally, subcu aneously o
in amuscula ly accina ed chickens we e p o ec ed, al hough no
p o ec ion was obse ed in conjunc i al sac accina ed chickens (Sheppa d
e al., 1998).
4.3. Adeno i us Vaccina ion in Swine
4.3.1. Classical Swine Fe e Vi us (CSFV)
Classical swine e e (CSF), also known as hog chole a, is a se ious and
con agious i al disease o pigs wi h a high mo ali y a e, di icul o
con ol in a eas o high pig o wild boa densi ies, being he mos
economically impo an disease o swine in a eas o in ensi e pig a ming.
Chap e I
16
Fo his eason, CSF is included in he A lis o in ec ious diseases o he
highes impo ance o in e na ional ade. Thus, i is highly ele an o
de elop e icacious accines o he con ol o CSFV in domes ic pigs and in
wild boa . P ophylac ic accina ion is s ill ca ied ou in many pa s o he
wo ld (Pa on and G eise -Wilke 2003). New accine de elopmen s ha e
included a numbe o di e en s a egies o deli e ing he majo en elope
glycop o ein, E2, agains which mos neu alizing an ibodies a e di ec ed
( an Oi scho 2003). Hammond e al. (2000), cons uc ed he i s PAd
exp essing he E2 p o ein and showed ha a single dose o 107
TCID50/animal in issue cul u e supe na an , when adminis e ed
subcu aneously, is su icien o comple ely p o ec pigs agains
subcu aneously challenge. La e , he same au ho s showed ha when he
challenge is adminis e ed o ally, only 60% o he animals we e p o ec ed
(Hammond e al., 2001b); mo e, ecen ly, i was shown ha pigs gi en wo
o al doses o 106 TCID50/animal o PAd we e comple ely p o ec ed om
he disease (Hammond e al., 2003).
4.3.2. Pseudo abies Vi us (P V)
P V is an alpha he pes i us which causes he economically impo an and
widesp ead Aujeszky’s disease (AJD) in pigs. P V is a highly neu o opic
i us causing ne ous and espi a o y complica ions in pigs, he na u al
hos , and in a a ie y o o he animal species. Vaccina ion agains AJD is
widely p ac ised wi h li e a enua ed o killed whole i us accines.
Howe e , neona al immuniza ion is o en limi ed in he p esence o
ma e nally-de i ed an ibody, which inhibi s he immune esponse agains
bo h accines (Ro h 1999). Recen ly, he use o Ad accines ca ying
indi idual P V genes we e cons uc ed as a sa e al e na i e. Glycop o eins
gD, gB and gC o P V we e chosen on he basis o hei ole in elici ing a
p o ec i e immune esponse agains i us in ec ion. I was shown ha
pigle s accina ed in amuscula ly wi h 108.6-109.6 TCID50/animal one day
In oduc ion
17
a e bi h, wi h eplica ion-compe en HAd5 ha bou ing hese h ee genes,
de eloped simila neu alizing an ibody esponses independen ly o he
p esence o absence o ma e nal an ibodies and we e pa ially p o ec ed
agains challenge 16 weeks la e (Mon eil e al., 2000). Fo pigs ha a e
slaugh e ed a ew mon hs a e bi h, one-sho accina ion a bi h could
p o ide p o ec ion o su icien du a ion. Finally, wo doses o 2 ml o
cla i ied issue cul u e supe na an con aining 105.4 TCID50 o PAd
exp essing he P V gD gene we e adminis e ed subcu aneously and showed
o p o ec pigs a e challenge. Pos -mo en, g oss lesions o pneumonia
we e ound in he lungs o pigs gi en a single dose o accine; he lungs o
pigs gi en wo doses we e ee om disease (Hammond e al., 2001a).
4.3.3. Foo and Mou h Disease Vi us (FMDV)
Foo and mou h disease (FMD) is a se e e, clinically acu e, esicula
disease o clo en-hoo ed animals including domes ica ed uminan s, pigs
and mo e han 70 wildli e species. Pigs a e ecognized as a signi ican
ac o in he sp ead o he disease since a single pig eleases as much
ae osol i us as 3000 ca le in a sho pe iod o ime (Alexande sen e al.,
2003; Thomson e al., 2003). The economic and social impac o FMD can
be ca as ophic when an ou b eak occu s in FMD- ee coun ies popula ed
wi h immunologically nai e animals. The cu en accine is a chemically
inac i a ed p epa a ion o concen a ed in ec ed cell cul u e supe na an .
Howe e , FMD- ee coun ies gene ally p ohibi i s use because o he lack
o an app o ed diagnos ic es ha can eliably dis inguish accina ed om
in ec ed animals. Mo eo e , cu en accines can induce a p o ec i e
esponse only a e app oxima ely 7 days pos accina ion; his is a c i ical
issue in disease- ee coun ies, whe e, in he case o FMD ou b eaks, a
apid con ol in p e en ing he sp ead o he disease is c ucial (see Doel
(2003) o a e iew). Thus, he e is a need o de elop disease con ol
s a egies elying on mo e apidly induced p o ec ion. May e al. (1999)

Chap e I
18
de eloped a eplica ion-de ec i e HAd5 ec o con aining he capsid
polypep ide P1 and he i al 3C p o ease coding egions, necessa y o
p ocessing P1 o he capsid p o eins VP0, VP3 and VP1, om he FMDV
s ain A12. They obse ed ha accina ed swine wi h 1×108 p u/animal in
PBS de eloped an ibodies agains FMDV s uc u al p o eins and an FMDV-
speci ic neu alizing an ibody esponse which seems o inc ease sligh ly by
boos ing he swine wi h a second inocula ion o 5×108 p u/animal in PBS a
4 weeks pos -ini ial accina ion (May e al., 2001). This accina ion
p o ocol was shown o o e a signi ican deg ee o p o ec ion o he pigs,
as i e o six pigs we e comple ely p o ec ed, while he emaining animal
had signi ican ly educed signs o disease. La e , Mo aes e al. (2002)
showed ha a single dose o 5×109 p u/animal in PBS o a eplica ion-
de ec i e HAd5 exp essing he P1 coding egion o FMDV s ain A24
comple ely p o ec ed pigs agains homologous challenge 7, 14 o 42 days
a e accina ion. Since e icacious accina ion is s ain dependen and he
in ec ion wi h one se o ype does no con e p o ec ion agains ano he ,
HAd5 bicis onic ec o accines ha e been de eloped (Wu e al., 2003),
dec easing he cos o mul i alen adeno i al FMD accines. Fo he
cons uc ion o hese ec o s, he P1 capsid coding egion o bo h A24 and
O1 s ains and he 3C p o ease coding egion o A12 s ain we e used.
Howe e , he neu alizing an ibody esponse a e accina ion wi h 2.5×109
p u/animal in PBS o he bicis onic ec o was conside ably lowe han
ha induced by a comme cial FMD accine o he mono alen Ad-A24
accine (Mo aes e al., 2002). Recen ly, a new s a egy based on he ac
ha FMDV is highly sensi i e o alpha/be a in e e on (IFN-/) ha e been
de eloped using a eplica ion-de ec i e HAd5 (Chinsanga am e al., 2003;
Mo aes e al., 2003). This s a egy has been shown o comple ely p o ec
pigs a e accina ion wi h 109 p u/animal when challenged 24 h la e wi h
i ulen FMDV.
In oduc ion
19
4.3.4. Po cine Respi a o y and Rep oduc i e Synd ome Vi us
(PRRSV)
PRRSV is he causa i e agen o an economically impo an pig disease,
wi h a wo ldwide dis ibu ion, cha ac e ized by ep oduc i e ailu e in
sows and espi a o y p oblems in unweaned and g owing pigs. Swine
mac ophage is he only cell ype known o suppo PRRSV eplica ion,
making comme cial p oduc ion impossible. Di ec con ac be ween
in ec ed and nai e pigs is he p edominan ou e o PRRSV ansmission.
Mo eo e , pneumonia caused by PRRSV in ec ion is mo e se e e in young
pigs compa ed o adul s and may be complica ed by concu en bac e ial
in ec ion (Rossow 1998). Gonin e al. (1999) obse ed ha PRRSV in ec ed
pigs p esen ci cula ing an ibodies esponsible o i al neu aliza ion
mainly di ec ed agains GP5, an en elope p o ein. Gagnon e al. (2003)
cons uc ed a eplica ion-de ec i e HAd5 exp essing he GP5 p o ein and
used his ecombinan i us o immunize pigs using wo in ade mal
injec ions wi h 5×108-1×109p u/animal in a mix u e con aining 100 L o
PBS and 100 L o poloxame SP1017 a 0.02%. I was obse ed ha
ollowing challenge gi en in anasally 14 days a e he boos e , pigs
p oduced high an ibody i e s o GP5 p o ein. Mo eo e , accina ed pigs
p esen ed speci ic immune memo y which, ollowing a subsequen PRRSV
in ec ion, esul ed in a apid clonal expansion o memo y cells o he
neu alizing epi opes o he au hen ic i al GP5 p o ein.
4.3.5. T ansmissible Gas oen e i is (Co ona) Vi us (TGEV)
TGEV in ec s he en e ic and espi a o y issues o newbo n pigle s
esul ing in mo ali ies app oaching 100%. The i us in ec s epi helial cells
and, in some cases, lung mac ophages (Ga wes 1988). The e a e se e al
comme cially a ailable TGEV accines, bo h inac i a ed and a enua ed;
hese do no ully p o ec pigle s. Se e al a emp s ha e been made o
Chap e I
20
de elop e icacious ecombinan TGEV accines (Tuboly and Nagy 2001).
The spike p o ein (S) was iden i ied as he majo induce o TGEV-
neu alizing an ibodies and i media es binding o TGEV o i s cellula
ecep o (Jimenez e al., 1986). Thus, HAd5 exp essing he S p o ein was
cons uc ed and used o s udy he induc ion o an ibodies p o iding
p o ec ion in swine (To es e al., 1995). I was obse ed ha po cine
se um, elici ed by 109 p u/animal in PBS o his ecombinan , when mixed
wi h a le hal dose o i us p io o adminis a ion o suscep ible pigs,
p e en ed he eplica ion o i ulen TGEV adminis e ed o ally as i us-
an ibody mix u es and ully p o ec ed swine om clinical signs and dea h.
Mo eo e , he used dose did no p oduce any clinical symp oms in any o
he mo e han 50 animals inocula ed up o 10 weeks a e inocula ion
(To es e al., 1996), sugges ing ha his ec o can be used as a li e
accine in swine wi hou seconda y complica ions associa ed wi h he
ec o . Howe e , a PAd ec o could be mo e e ec i e han HAd. Thus,
Tuboly and Nagy (2001) cons uc ed a PAd5 exp essing he TGEV p o ein.
I was obse ed ha a single o al dose o 5×106 p u/animal o he
ecombinan i us was su icien o induce bo h a sys emic and a local
humo al immune esponse (Tuboly and Nagy 2001). Un o una ely challenge
expe imen s we e no ca ied ou .
4.3.6. Swine In luenza Vi us (SIV)
SIV is a widesp ead and impo an pa hogen in species as di e se as
poul y, swine, ma ine mammals and humans. In pigs, in luenza can occu
ei he as an enzoo ic p oblem in a he d o , mo e commonly, as explosi e
ou b eaks o acu e espi a o y disease. Al hough a ely a al, swine
in luenza can be o subs an ial economic impac ( an Ree h and Nauwynck
2000). In addi ion, he e is g owing conce n o he po en ial o syne gis ic
in ec ions wi h in luenza and PRRSV. Beyond he impac o in luenza o
he swine indus y, pigs a e also e y impo an in he global ecology o
In oduc ion
21
in luenza A i uses in humans (Zhou e al., 1999). SIV accines ha a e
comme cially-a ailable a e inac i a ed, whole- i us o subuni accines.
While hese accines may dec ease he incidence and se e i y o clinical
disease, hey do no consis en ly p o ide comple e p o ec ion om i us
in ec ion. Two eplica ion-de ec i e HAd5 we e de eloped as po en ial
accines agains SIV: HAd5 exp essing he in luenza i us H3
haemagglu inin (HA) (Tang e al., 2002; Wesley e al., 2004) inducing
p edomina ely a sub ype-speci ic humu al immune esponse (Macklin e al.,
1998); and HAd5 exp essing he nucleop o ein (NP) (Wesley e al., 2004), a
g oup-speci ic s imula ing cy o oxic T lymphocy es o c oss- eac i e
immuni y o all in luenza A sub ypes (Ulme e al., 1998). I was obse ed
ha he immuniza ion o mice wi h 5×108 TCID50/animal and pigs wi h
2×1010 TCID50/animal in PBS wi h he i s ecombinan , desc ibed abo e,
de eloped high le els o i us-speci ic hemagglu ina ion-inhibi ion an ibody
o SIV by 4 weeks pos accina ion and he animals we e pa ially
p o ec ed. On he o he hand, pigs accina ed wi h 2×1010 TCID50/ml in PBS
o bo h ecombinan i uses in a mix u e we e comple ely p o ec ed.
4.4. Adeno i us Vaccina ion in Ca le
4.4.1. Bo ine Vi al Dia hea Vi us (BVDV)
BVDV is esponsible o educed milk p oduc ion, educed ep oduc i e
pe o mance and g ow h e a da ion. In addi ion, acu e in ec ion in adul
ca le, congeni al de ec s and inc eased neona al mo ali y a e also clinical
mani es a ions o BVDV in ec ion (Goens 2002). Finally, i was epo ed
ha BVDV may play an indi ec ole in immunosupp ession (Wellenbe g e
al., 2002). Cu en ly, inac i a ed and modi ied-li e accines a e used;
howe e , bo h ypes o accines ha e signi ican sho comings. A HAd5
exp essing he nucleocapsid C p o ein (p14), ha is highly conse ed
among many di e en pes i i uses, o which BVDV belongs, was
Chap e I
28
ba ch mode p o iding he easies way o p oceed as no ex a eeding is
equi ed and he isk o con amina ion is lowe ed gi en he simplici y o
ope a ion; ii) ed-ba ch mode, easy o ope a e and eadily scalable, is
employed o ex end cul u e li e ime by supplemen ing limi ing nu ien s o
educing he accumula ion o oxic me aboli es; and iii) pe usion mode,
consis ing in cell e en ion a a ela i ely high concen a ion inside he
bio eac o , while esh nu ien supply and me aboli e emo al akes place
(see Kamen and Hen y (2004) o a e iew).
Me hodologies o p oduc ion o concen a ed AdV a low cos a e
manda o y as he ma ke needs o Ad a e inc easing. Al hough Ad has he
ad an age o be p oduced a high i e s (1010-1011 p u/ml) (Babiuk and
Tikoo 2000), o ob ain a good immune esponse in a la ge p opo ion o
ea ed animals, pa icula ly o mucosal accina ion, la ge doses and hus
cul u e olumes a e equi ed. Fu he p ocess de elopmen aiming a
highe yields o p oduc is clea ly necessa y. Imp o emen s in olume ic
p oduc ion can be achie ed by inc easing he cell densi y a which cells can
be in ec ed wi hou lowe ing he speci ic yield o he p oduc ; howe e ,
p oduc ion o AdV ha main ain a high speci ic yield in ba ch ope a ion is
limi ed o cell densi ies in he ange o 1×106 cell/ml; se e al app oaches
ha e been made in o de o o e come his so called “cell densi y e ec ”
(Nadeau and Kamen 2003): Ga nie e al. (1994) demons a ed ha
medium eplacemen a in ec ion and he addi ion o glucose a 24 hou s
pos in ec ion (hpi) oge he wi h pe iodical pH adjus men s, allowed a
sus ained maximum speci ic p oduc i i y a 1.6106 cell/ml whe eas
Nadeau e al. (1996), u he imp o ed his s a egy by also adding
essen ial amino acids a 24 hpi he eby s abilising olume ic p oduc i i y
a cell densi ies abo e 2106 and below 3106 cell/ml. Such esul s hin a
he exis ence o subs a e limi a ion and/o byp oduc inhibi ion a high
cell densi ies. A p oduc ion scale, medium exchange will inc ease he cos
o he inal p oduc , which is e en mo e c i ical when his is o be used in
he e e ina y ield. Pe usion mode ope a ion has been a emp ed as a

In oduc ion
29
means o con ol he cul u e en i onmen and emo e oxic byp oduc s
(Hen y e al., 2004; Kamen and Hen y 2004); ne e heless, he cell speci ic
p oduc i i y could only be main ained by in ec ing cells a densi ies up o
3106 cell/ml using high pe usions a es o 2 eac o olumes pe day, a 2
days pos in ec ion, a e y cos ly p oposi ion (Kamen and Hen y 2004).
The impo ance o in ec ion kine ics on Ad p oduc ion and he signi icance
o a iables such as Mul iplici y O In ec ion (MOI) and ha es ing ime in
p ocess op imisa ion is also manda o y o inc ease p oduc ion yields, o
a oid apid deple ion o cos ly and ce i ied mas e i us banks as well as
o ensu e ha he in ec ion kine ics is ep oducible be ween di e en
p oduc ion scales. Mo eo e , he use o low MOI a la ge scale would be
p e e en ial since an in e media e s ep o i us inoculum p oduc ion would
be a oided (Annex A).
Finally, e e ina y p oduc s should be pu i ied wi h a minimal numbe o
s eps and he uni ope a ions employed should be simple and non-
expensi e. T adi ionally, labo a o y pu i ica ion o Ad was achie ed using
wo ounds o cesium chlo ide (CsCl) densi y g adien ul acen i uga ion.
Howe e , CsCl densi y g adien me hod is no scaleable. Despi e he ac
ha ch oma og aphic pu i ica ion is an expensi e me hod, ion exchange,
hyd ophobic in e ac ion, me al chela e and size-exclusion ch oma og aphy
ha e been e alua ed o cap u e and pu i ica ion o HAd5 (Huyghe e al.,
1995). Kamen and Hen y (2004) de eloped a me hod o gene he apy
applica ion consis ing o : i) ha es o in ec ed cells by con inuous
cen i uga ion, ii) cell lysis by osmo ic shock, iii) DNAse ea men wi h
cen i ugal/condi ioning, i ) il a ion, ) anion-exhange ch oma og aphy,
i) ul a il a ion/concen a ion and ii) size exclusion ch oma og aphy.
This p o ocol allows la ge scale pu i ica ion o AdV wi h pu i y compa able
o he CsCl g adien me hod; howe e , i inc eases he inal p oduc p ice,
well beyond he each o e e ina y u ilisa ion. In ogen de eloped a
me hod consis ing o a single ion ch oma og aphy un, a e
concen a ion/dia il a ion and nuclease ea men , wi h a o al eco e y
Chap e I
30
o he i us p oduc o 70% (Zhang e al., 2001). Also, a p o ocol o pu i y
HAd5 om he bulk ha es ed di ec ly om he bio eac o a e lysis
(wi hou a concen a ion s ep) was de eloped using a single ion exchange
ch oma og aphic s ep ollowed by ul a il a ion wi h a inal yield o 32%
achie ed in less han one wo king day wi h a minimal amoun o sample
manipula ion (Peixo o e al., 2005). This p ocess p esen ed he ad an age
o cap u ing he i al pa icles di ec ly om cellula ex ac s and o
accoun o he signi ican amoun s (25-80%) o AdV ha a e p esen in he
cul u e medium a ha es ime due o ea ly cell lysis (Schagen e al.,
2000).
6. SCOPE OF THE THESIS
One o he mains goals o a comme cial AdV cul i a ion p ocess o gene
he apy o accine applica ions is o maximise he i us olume ic
p oduc i i y, de ined as uni s o i al in ec i e pa icles, pe cul u e
olume pe uni ime. Since one can in ec a di e en cell concen a ions,
he cell speci ic p oduc i i y, de ined as i al in ec i e pa icles p oduced
pe cell and uni ime, is a pa allel concep ; ideally, ope a ing a he
highes possible cell speci ic p oduc i i y ob ained a he highes cell
concen a ion a in ec ion would maximize he olume ic p oduc i i y o
he p ocess bu quali y o he p oduced AdV is o pa amoun impo ance.
This hesis has a emp ed o imp o e he cu en manu ac u ing p ocess o
AdV p oduc ion by pe o ming in ec ion a high cell densi ies main aining
he cell speci ic p oduc i i y. To achie e his, he bo lenecks in cell
p oduc i i y ha e been s udied as well as he pa ame e s ha may a ec
he ec o p oduc ion a high cell densi ies. The cell densi y a in ec ion is
a e y impo an pa ame e as i s ongly impac s he AdV olume ic
p oduc i i y. Howe e , in simple ba ch-mode ope a ions, a ela i ely
na ow ange o op imal cell densi y a in ec ion o 1×106 cell/ml has been
consis en ly epo ed. The easons o he d op in pe cell p oduc i i y a
In oduc ion
31
highe cell densi ies a e no cu en ly known, as medium o mula ions
allow maximum cell g ow hs eaching 8×106 cell/ml. The e o e mo e
esea ch is needed o unde s and and o e come his phenomenon o enable
simple ba ch p ocesses o each highe p oduc i i ies. In o de o design a
a ional p oduc ion p ocess ha allow he maximisa ion o he AdV yields
a high cell densi ies, ou di e en bio eac ion app oaches we e explo ed
in his hesis: i) media exchange, ii) e eed s a egy, iii) use o non-
ammoniagenic medium and i ) cell cycle synch onisa ion. The knowledge
gained in his scien i ic excu sion is in eg a ed in o an imp o ed p ocess.
7. ACKNOWLEDGEMENTS
The au ho s acknowledge and app ecia e he inancial suppo ecei ed
om he Eu opean Commission (P ojec RP/PPR ORALVAC ICA4-CT-2000-
30027) and om Fundação pa a a Ciência e Tecnologia – Po ugal (P ojec
POCTI/BIO/46515/2002) and s uden g an (SFRH/BD/10614/2002)).
Chap e I
32
8. REFERENCES
Alexande sen, S., Zhang, Z., Donaldson, A.I. and Ga land, A.J., 2003. The
pa hogenesis and diagnosis o oo -and-mou h disease. J Comp Pa hol 129, 1-36.
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In oduc ion
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Chap e I
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In oduc ion
35
Hammond, J.M., McCoy, R.J., Jansen, E.S., Mo issy, C.J., Hodgson, A.L. and
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Chap e I
36
Jooss, K., E l, H.C. and Wilson, J.M., 1998. Cy o oxic T-lymphocy e a ge
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Macklin, M.D., McCabe, D., McG ego , M.W., Neumann, V., Meye , T., Callan, R.,
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May , G.A., Chinsanga am, J. and G ubman, M.J., 1999. De elopmen o
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coding egions o oo -and-mou h disease i us as a accine candida e. Vi ology
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In oduc ion
37
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dependen E1-posi i e pa icles. Hum Gene The 13, 909-20.
Mu wi i, G., Ba eman, C., Baca-Es ada, M.E., Snide , M. and G iebel, P., 2000.
Induc ion o immune esponses in newbo n lambs ollowing en e ic immuniza ion
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adeno i us-speci ic immuni y on immune esponses induced by ecombinan

Cell Densi y E ec
45
Two di e en se um- ee media and osmolali y e ec upon human 293
cell g ow h and adeno i us p oduc ion
Fe ei a T.B., Fe ei a A.L., Ca ondo M.J.T., Al es P.M. (2005) Bio echnol.
Le ., 27:1809-1813.
Chap e II
46
Abs ac
Adeno i uses a e p omising ec o s o gene he apy and accina ion
p o ocols. Consequen ly, he ma ke equi emen s o adeno i us a e
inc easing, d i ing he sea ch o new me hodologies o la ge-scale
p oduc ion o concen a ed ec o s wi h wa an ed pu i y and e icacy, in
a cos -e ec i e way. Ne e heless, he p oduc ion o adeno i us is
cu en ly limi ed by he so-called “cell densi y e ec ”, i.e., a d op in cell
speci ic p oduc i i y concomi an wi h inc eased cell concen a ion a
in ec ion. In his s udy, wo di e en se um- ee cul u e media (CD 293
and EX-Cell) a e e alua ed in hei e ec on human 293 cells g ow h and
adeno i us p oduc ion a cell densi ies highe han 1106 cells/ml. The
esul s show ha EX-Cell was he be e medium o cell g ow h. Al hough
adeno i us p oduc ion was equi alen in bo h CD 293 and EX-Cell media
when he in ec ion was pe o med a 1106 cells/ml, a 3106 cells/ml CD
293 p o ed o be he be e medium. This esul was ela ed wi h he high
ammonia con en in EX-Cell medium a he highes cell concen a ion a
in ec ion. Besides his, he la ge-scale p oduc ion o hese ec o s a high
cell densi ies o en equi es e eed s a egies, which inc ease medium
osmolali y. While a nega i e e ec on cell g ow h was obse ed wi h
inc easing osmolali ies, adeno i us p oduc i i y was only a ec ed o
osmolali ies highe han 430 mOsm.
Cell Densi y E ec
47
CONTENTS
1. INTRODUCTION_________________________________________ 48
2. MATERIALS AND METHODS ________________________________ 49
2.1. Cell line and cul u e condi ions ________________________ 49
2.2. In ec ion in shake lasks ______________________________ 49
2.3. Vi us cul u e samples p epa a ion ______________________ 50
2.4. Analy ical Me hods __________________________________ 50
3. RESULTS AND DISCUSSION ________________________________ 50
3.1. E ec o media on cell g ow h _________________________ 50
3.2. E ec o media on AdV p oduc i i y_____________________ 51
3.3. E ec o osmolali y inc ease on cell g ow h and AdV p oduc i i y
_____________________________________________________ 53
4. ACKNOWLEDGEMENT ____________________________________ 56
5. REFERENCES___________________________________________ 57
Chap e II
48
1. INTRODUCTION
Recombinan adeno i uses ha e been he ec o s o choice o gene
he apy and accina ion due o hei high e iciency o gene ans e in a
b oad spec um o cell ypes (Rea e al., 1999; Moun ain, 2000). The e o e,
ma ke needs o adeno i us ec o s (AdV) a e cons an ly inc easing
causing a high demand o me hodologies o he la ge-scale p oduc ion o
concen a ed ec o s wi h wa an ed le els o pu i y and e icacy a a low
cos (Fe ei a e al., 2005a). Di e en app oaches o he imp o emen o
p oduc ion and pu i ica ion p ocesses o hese i al ec o s ha e mos ly
used se um- ee media (Nadeau e al., 1996; Co e e al., 1997; Co e e al.,
1998). Se um- ee media o mula ions a e, howe e , mo e expensi e han
he adi ional ones wi h se um supplemen a ion and also he cell speci ic
p oduc i i y dec eases sha ply wi h inc eased cell concen a ion a
in ec ion (CCI) abo e 1×106 cell/ml, he so called “cell densi y e ec ”
(Nadeau and Kamen 2003; Kamen and Hen y 2004). The easons o such an
e ec a e no e y well unde s ood bu he lack o an essen ial nu ien o
g ow h ac o s o high concen a ion o me abolic inhibi o y p oduc s,
p esen a high cell densi ies, a e he mos p obable causes. Recen ly,
se e al comme cial se um- ee media ha e become a ailable; hus, one o
he goals o his s udy was o e alua e he e ec o wo di e en
comme cially a ailable se um- ee media on human 293 cell g ow h and
AdV p oduc i i y a high cell densi ies.
Besides cul u e media composi ion and he accumula ion o inhibi o y
byp oduc s, se e al o he ac o s ha e been epo ed o in luence cell
g ow h and AdV p oduc i i y, namely he ec o i sel , he cell line, he
pH, pCO2 and empe a u e (Eloi and Adam 1995; Iye e al., 1999; Ja don
and Ga nie 2003; Nadeau and Kamen 2003). Due o he common need o
implemen e eeding s a egies o AdV p oduc ion, i is pe inen o
de e mine i he consequen inc ease in osmolali y has an in luence on cell
g ow h and AdV p oduc i i y. Only one s udy has epo ed he in luence o
Cell Densi y E ec
49
osmolali y on p o ein p oduc ion by human 293 cells (Nadeau e al., 1996),
esul ing in inc eased p oduc ions a high osmolali ies (500 mOsm).
Ne e heless, he e ec o osmolali y on i al p oduc i i y has no been
assessed ye , hus his s udy epo s e ec o osmolali y upon human 293
cell g ow h and AdV p oduc i i y.
2. MATERIALS AND METHODS
2.1. Cell line and cul u e condi ions
Suspension-adap ed human 293 cells (ATCC-CRL-1573) we e g own in 125
ml E lenmeye laks, wi h 40 ml medium in wo di e en se um ee
media: CD 293, a p o ein- ee medium om In i ogen and EX-Cell, an
animal-p o ein ee medium om JRH, all supplemen ed wi h 6 mM
glu amine om In i ogen. Cells we e incuba ed a 37 ºC in a humidi ied
a mosphe e o 8 and 10 % o CO2 in ai , espec i ely (in o de o ob ain an
equal pH in each medium), wi h a cell inoculum o 0.25×106 cell/ml.
Rega ding he osmolali y s udies, i e di e en osmolali ies we e
e alua ed: 230, 330, 380, 430 and 480 mOsm. The osmolali y o he
medium was inc eased by addi ion o a 1 M NaCl.
2.2. In ec ion in shake lasks
A eplica ion-de ec i e AdV de i ed om ype 5 AdV was kindly p o ided by
D . Tom Ba e (Ins i u e o Animal Heal h-Pi b igh , UK). In ec ion was
made when he cells eached a concen a ion o 1×106 cell/ml using a
mul iplici y o in ec ion (MOI) o 10 (see annex A). In ec ion was also made
when cells eached a concen a ion o 2×106 cell/ml and 3×106 cell/ml o
he s udies on media e ec , main aining he same MOI. In ec ed human
293 cells we e ha es ed a 48 h pos in ec ion (hpi), de e mined as he
op imal ha es ime (Annex A).

Chap e II
50
2.3. Vi us cul u e samples p epa a ion
In ec ed human 293 cells we e ha es ed a 48 hpi and cen i uged a 1000
g o 10 min a 4 ºC; he esul ing cell supe na an was dis ibu ed in o
small aliquo s and s o ed a -85 ºC o he e alua ion o ex acellula i us;
he esul ing cell pelle was esuspended in a known olume o lysing
bu e (T is/HCl 10 mM, pH 8.0, 2mM MgCl2 and 0.1% T i on-X 100) and he
cells we e dis up ed by 1 minu e o ex. Cell deb is was emo ed by
cen i uga ion a 3000 g o 10 min a 4 ºC and he esul ing cell lysa e
supe na an was dis ibu ed in o small aliquo s and s o ed a –85 ºC o
quan i ica ion o in acellula i uses con en .
2.4. Analy ical Me hods
Cell concen a ion and iabili y we e de e mined by coun ing he cells on a
haemacy ome e using he T ypan Blue dye exclusion me hod.
AdV i a ion was pe o med by he end-poin dilu ion me hod using 96 well
pla es and human 293 cells. In ec ious pa icles (ip) in 50 % issue cul u e
in ec i e dose (TCID50) we e de e mined acco ding o he s a is ical
me hod o Spea man-Ka be (Da ling e al., 1998).
Ammonia was quan i ied enzyma ically using a UV- es Ca . No.
1112732035 (Boeh inge Mannheim, R-Biopha m AG)
Osmolali y was measu ed in a Digi al Mic o Osmome e Type 5R om
He mann Roebling MESSTECHNIK, Ge many.
3. RESULTS AND DISCUSSION
3.1. E ec o media on cell g ow h
In o de o ob ain high cell yields o AdV p oduc ion, wo di e en se um-
ee media we e es ed in hei abili y o g ow human 293 cells. As shown
Cell Densi y E ec
51
in Figu e 2.1, EX-Cell allowed a maximum cell numbe o 7.5×106 cell/ml,
while he maximum cell numbe ob ained in CD 293 was app oxima ely
4×106 cell/ml, a ained a simila speci ic g ow h a es (0.018 h-1 and 0.019
h-1 o CD 293 and EX-Cell, espec i ely). Despi e he ac ha EX-Cell has a
highe glucose concen a ion (30 mM) han CD 293 (25 mM), he main
di e ence obse ed be ween he wo media composi ion is in he amino
acids concen a ion. By ollowing amino acid consump ion i was seen ha
h eonine was he unique amino acid ha was comple ely consumed when
cells eached 4×106 cell/ml in CD 293 (da a no shown). Fu he mo e, EX-
Cell con ains a concen a ion o h eonine 10 imes highe han CD 293,
and he e eeding o his amino acid in CD 293 led o an inc ease in
maximum iable cell concen a ion o 5.5×106 cell/ml (da a no shown).
Time (days)
Viable cells (×10
6
cell/ml)
3
4
5
6
7
8
6
0
1
2
0 1 2 3 4 5 7 8 9 10 11
Time (days)
Viable cells (×10
6
cell/ml)
3
4
5
6
7
8
6
0
1
2
0 1 2 3 4 5 7 8 9 10 11
Figu e 2.1. G ow h cu es o human 293 cells ob ained in wo di e en cul u e
media: (o) CD 293 and (□) EX-Cell.
3.2. E ec o media on AdV p oduc i i y
Since EX-Cell medium allowed he highe maximum cell densi y, he e ec
upon AdV p oduc i i y a high CCI was e alua ed and compa ed wi h CD
293 medium. As shown in Figu e 2.2, when a CCI o 1×106 cell/ml was used,
Chap e II
52
bo h cul u e media we e simila in hei abili y o “suppo ” AdV
p oduc ion. The speci ic p oduc i i y in human 293 cells using CD 293
medium was no signi ican ly a ec ed when a CCI o 2×106 cell/ml was
used, while a 10 old dec ease was obse ed when EX-Cell medium was
used. Fo a CCI o 3×106 cell/ml no e ec i e AdV p oduc ion was obse ed
o EX-Cell, and o CD 293 a 1 log dec ease (as compa ed wi h CCI o 1×106
cell/ml) was obse ed, con i ming he CD 293 medium as a good choice o
AdV p oduc ion a high cell densi ies (Fe ei a e al., 2005b).
CCI 1 CCI 2 CCI 3
1E+00
1E+01
1E+02
1E+03
1E+04
CCI 1 CCI 2 CCI 3
ip
/Cell
CCI 1 CCI 2 CCI 3
1E+00
1E+01
1E+02
1E+03
1E+04
CCI 1 CCI 2 CCI 3
ip
/Cell
Figu e 2.2. Cell speci ic p oduc i i y in CD 293 (■) and EX-Cell (□) a CCIs o 1, 2
and 3×106 cell/ml. E o ba s ep esen he p opaga ed e o conside ing one SD o
quad uplica e sample assay o wo samples.
The dec ease in cell speci ic p oduc i i y ob ained a a CCI o 3×106 cell/ml
in CD 293 and EX-Cell media migh be due o he lack o an essen ial
nu ien o g ow h ac o s o o he byp oduc s accumula ion. As s a ed in
Chap e III, Pa 1, glu amine, h eonine and glucose a e limi ing nu ien s
in CD 293 medium a CCI o 3×106 cell/ml, al hough no imp o emen on
AdV p oduc i i y was achie ed a e he addi ion o hese nu ien s a he
ime o in ec ion; on he o he hand, ammonia was a an inhibi o y
concen a ion o in ec ion a his CCI. In his p esen s udy, ollowing
ammonia p oduc ion o bo h media, i was obse ed ha a 3×106 cell/ml
Cell Densi y E ec
53
ammonia concen a ion was 1.8 imes highe in EX-Cell medium han in CD
293 (Figu e 2.3), suppo ing ha ammonia is one o he main con ibu o
o he “cell densi y e ec ”.
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Viable cells (10
6
Cell/ml)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0.0
0 1 2 3 4 5 6 7 8
Time (h)
Ammonia (mM)
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Viable cells (10
6
Cell/ml)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0.0
0 1 2 3 4 5 6 7 8
Time (h)
Ammonia (mM)
Figu e 2.3. Ammonia p oduc ion ( ull lines) du ing cell g ow h (b oken lines) in CD
293 (●, ○) and in EX-Cell (■, □).
3.3. E ec o osmolali y inc ease on cell g ow h and AdV p oduc i i y
The e ec o inc easing osmolali y on human 293 cell g ow h was
e alua ed by inc easing i o 330, 380, 430 and 480 mOsm om o iginal
alues o 230 mOsm a he inocula ion ime and when he cells eached a
concen a ion o 1×106 cell/ml (concen a ion whe e he cell speci ic
p oduc i i y is maximal). As shown in Table 2.1, dec eases on speci ic
g ow h a e and maximum iable cell concen a ion we e obse ed when
he osmolali y was inc eased a he innocula ion ime, leading o g ow h
inhibi ion a he maximum osmolali y s udied (480 mOsm), whe eas
osmolali y inc eases a 1×106 cell/ml had a lesse e ec upon bo h speci ic
g ow h a e and maximum iable cell concen a ion.

Re eed s a egies and non-ammoniagenic medium
61
PART 1
E ec o e eed s a egies and non-ammoniagenic medium on
adeno i us p oduc ion a high cell densi ies
Fe ei a T.B., Fe ei a A.L., Ca ondo M.J.T., Al es P.M. (2005) J.
Bio echnol., 119:272-280.
Chap e III.1
62
Abs ac
Recombinan adeno i uses became one o he ec o s o choice o deli e y
and exp ession o o eign p o eins o gene he apy and accina ion
pu poses. Ne e heless, he p oduc ion o adeno i us is cu en ly limi ed
by he so-called “cell densi y e ec ”, i.e., a d op in cell speci ic
p oduc i i y concomi an wi h inc eased cell concen a ion a in ec ion
(CCI).
This wo k desc ibes he cha ac e isa ion and op imisa ion o he in ec ion
p ocess in o de o imp o e ecombinan adeno i us ype 5 yields a high
cell densi ies. Fo ha pu pose, 293 cells adap ed o suspension we e
g own in 2 L bio eac o s and in ec ed a di e en cell concen a ions,
using di e en e eed s a egies, while e alua ing cell me abolism. The
consump ion o amino acids is enhanced du ing in ec ion, al hough no
amino acid limi a ion was de ec ed o cells in ec ed a concen a ions in
he ange o 2106 cell/ml, o which he highes olume ic p oduc i i y
was ob ained in ba ch mode. Con e sely, in ec ing a cell concen a ions in
he ange o 3106 cell/ml led o comple e deple ion o glucose, glu amine
and h eonine be o e he op imal ha es ing ime, a signi ican dec ease in
olume ic p oduc i i y being obse ed; he e ec o amino acids and
glucose addi ion a in ec ion ime on cell speci ic and olume ic
p oduc i i y o adeno i us was assessed, no imp o emen on adeno i us
p oduc ion being achie ed. The e ec o ammonia, p esen in high
concen a ions a 3106 cell/ml, was e alua ed and seem o be
de imen al; an 1.8 old inc ease on adeno i us olume ic p oduc i i y
was ob ained o in ec ions pe o med a 3106 cell/ml when non-
ammoniagenic medium was used.
Re eed s a egies and non-ammoniagenic medium
63
CONTENTS
1. INTRODUCTION_________________________________________ 64
2. MATERIALS AND METHODS ________________________________ 66
2.1. Cell line and medium ________________________________ 66
2.2. In ec ion in shake lasks ______________________________ 66
2.3. S i ed ank s udies__________________________________ 66
2.4. Vi us cul u e samples p epa a ion ______________________ 67
2.5. Analy ical Me hods __________________________________ 67
3. RESULTS AND DISCUSSION ________________________________ 68
3.1. E ec o CCI on AdV p oduc ion ________________________ 68
3.2. Cell me abolism e alua ion a di e en CCIs ______________ 69
3.3. AdV p oduc ion wi h a e eed s a egy___________________ 72
3.4. AdV p oduc ion in non-ammoniagenic media ______________ 73
4. CONCLUSIONS__________________________________________ 76
5. ACKNOWLEDGEMENT ____________________________________ 77
6. REFERENCES___________________________________________ 78
Chap e III.1
64
1. INTRODUCTION
Deli e y o genes o soma ic issue o he p e en ion and ea men o a
wide ange o gene ic and acqui ed diseases is a p omising ou e o
he apy. The added genes can p o ide a missing unc ion, modula e he
immune esponse o ini ia e cell suicide in he p esence o ce ain d ugs.
Since ecombinan adeno i uses (AdV) a e highly e icien a gene ans e
o a b oad spec um o cell ypes and species hey became one o he
ec o s o choice o gene deli e y and exp ession o o eign p o eins in
gene he apy and accina ion (Rea e al., 1999; Moun ain 2000).
Consequen ly, he ma ke equi emen s o AdV a e inc easing, d i ing he
sea ch o new me hodologies o la ge scale p oduc ion o concen a ed
ec o s wi h wa an ed pu i y and e icacy.
Di e en app oaches o he imp o emen o p oduc ion and pu i ica ion
p ocesses o hese i al ec o s ha e been epo ed (Huyghe e al., 1995;
Nadeau e al., 1996; Iye e al., 1999; Blanche e al., 2000). Fac o s such
as he cons uc ion o he ec o i sel , he hos cell line and he cul u e
medium can a ec AdV p oduc ion a es (Eloi and Adam 1995; Iye e al.,
1999; Nadeau and Kamen 2003). E ec s o pH, pCO2 and empe a u e on
AdV p oduc ion ha e also been epo ed (Xie e al., 2002; Ja don and
Ga nie 2003). Cu en ly, AdV p oduc ion a high cell speci ic p oduc i i y
is limi ed o low cell densi ies, in he ange o 1106 cell/ml (Kamen and
Hen y 2004). This “cell densi y e ec ” has been conside ed by se e al
au ho s as a consequence o nu ien limi a ions o accumula ion o
byp oduc s. The unde s anding o hese limi ing ac o s will con ibu e o
design a obus and cheap p ocess o AdV p oduc ion a high cell densi ies,
by adding he essen ial nu ien s o using a new medium o mula ion ha
allows he in ec ion a highe cell densi ies main aining he speci ic cell
p oduc i i y. Al hough me abolic lux analysis has been used o
cha ac e ize he e ec upon cell me abolism o a ious en i onmen al
condi ions he na u e o he ac o s limi ing AdV p oduc i i ies a high cell
Re eed s a egies and non-ammoniagenic medium
65
densi ies emains unknown (Nadeau e al., 2000a; Nadeau e al., 2000b;
Nadeau e al., 2002).
Di e en s a egies o imp o emen ha e been p oposed: Ga nie e al.
(1994) demons a ed ha medium eplacemen a 0 hou s pos in ec ion
(hpi) and he addi ion o glucose a 24 hpi oge he wi h pe iodical pH
adjus men s, allowed a sus ained maximum cell speci ic p oduc i i y a
1.6106 cell/ml whe eas, Nadeau e al. (1996) u he imp o ed his
s a egy by also adding essen ial amino acids a 24 hpi he eby s abilising
AdV olume ic p oduc i i y a cell densi ies jus abo e 2106 cell/ml. A
p oduc ion scale, medium exchange will inc ease he cos o he inal
p oduc , which is e en mo e c i ical when he inal p oduc is o be used in
he e e ina y ield. Pe usion mode ope a ion has been a emp ed as a
means o con ol he cul u e en i onmen and emo e oxic byp oduc s
(Hen y e al., 2004; Kamen and Hen y 2004); ne e heless, he cell speci ic
p oduc i i y could only be main ained by in ec ing cells a densi ies up o
3106 cell/ml using high pe usions a es o 2 eac o olumes pe day, a 2
days pos in ec ion, a e y cos ly p oposi ion.
The accumula ion o ammonia and lac a e, majo p oduc s om glu amine
and glucose me abolism, has been demons a ed o diminish mammalian
cell g ow h and in luence cell me abolism and p o ein p oduc i i y (C uz e
al., 2000). A emp s ha e been made o con ol cell me abolism and
educe he p oduc ion o hese me aboli es, such as: i) main aining low
concen a ions o glucose and glu amine o shi cell me abolism owa ds
mo e e icien s a es (C uz e al., 1999), o ii) adap ing he cells o g ow in
media wi h o he ca bon and ni ogen sou ces like galac ose and glu ama e
(Al ami ano e al., 2000). Speci ically, o 293 cells o en u ilised o AdV
p oduc ion, lac a e a 20 mM educed cell iabili y o 50% wi h a much
educed e ec on p oduc , whe eas ammonia a 1 mM al eady inhibi ed
cell g ow h; unde hese condi ions he e ec s upon AdV p oduc ion we e
no iden i ied (Nadeau e al., 1996).
In his wo k, ac o s limi ing he AdV p oduc ion a high cell densi ies we e

Chap e III.1
66
in es iga ed. Re eed s a egies and he adap a ion o non-ammoniagenic
media we e e alua ed in o de o unde s and he me abolic equi emen s
when in ec ion is done a high cell densi ies and o o e come he so called
"cell densi y e ec ", hus pe mi ing he achie emen o highe olume ic
p oduc i i ies o AdV. A i s gene a ion AdV ec o was employed.
2. MATERIALS AND METHODS
2.1. Cell line and medium
293 cells, pu chased om ATCC (ATCC-CRL-1573), we e adap ed o
suspension and g own in comme cially a ailable se um and p o ein ee
medium, CD 293, supplemen ed wi h 4 mM o glu amine (all om
In i ogen, Glasgow, UK) a a humidi ied a mosphe e o 8% CO2 in ai a
37ºC in shake lasks (co ning, NY). Cells we e ou inely p opaga ed wice a
week wi h an inoculum o 0.5×106 cell/ml.
2.2. In ec ion in shake lasks
In ec ion was pe o med in 125 ml shake lasks wi h inal olumes o 40 ml
in a humidi ied a mosphe e o 8% CO2 in ai a 37ºC. All he in ec ions we e
done using a mul iplici y o in ec ion (MOI) o 10 and AdV ha es ed a 48
hpi (see Annex A).
2.3. S i ed ank s udies
Bio eac ion s udies we e pe o med in a 2 L bio eac o (B aun, Melsungen,
Ge many). The agi a ion a e was main ained a 110 pm (NRe = 6800); pH
was con olled a 7.2 by ae a ion wi h a CO2 gas-mix u e and NaOH 0.2 M;
he dissol ed oxygen was con olled a 80% ai sa u a ion. All he in ec ions
we e done using a MOI o 10 and AdV ha es ed a 48 hpi (see Annex A). Fo
Re eed s a egies and non-ammoniagenic medium
67
he ed-ba ch ope a ion mode, h eonine, glu ama e, amino acids cock ail
(RPMI 1640 50) (all om Sigma-Ald ich, S . Louis, MO), glu amine and
glucose (Me ck, Da ms ad , Ge many) solu ions we e used.
2.4. Vi us cul u e samples p epa a ion
A eplica ion-de ec i e AdV de i ed om ype 5 AdV was kindly p o ided by
D . Tom Ba e (IAH-Pi b igh , UK). In ec ed 293 cells we e ha es ed a 48
hpi and cen i uged a 1000g o 10 min. a 4ºC. The esul ing cell
supe na an was dis ibu ed in o small aliquo s and s o ed a -85ºC o he
e alua ion o ex acellula i us. The esul ing cell pelle was esuspended
in a known olume o T is bu e 10 mM, pH 8.0 supplemen ed wi h 2 mM
MgCl2 and 0.1% T i on. Cells we e dis up ed by o exing o 1 minu e and
cell deb is emo ed by cen i uga ion a 3000g o 10 min. a 4ºC. The
esul ing cell lysa e supe na an was dispensed in o small aliquo s and
s o ed a -85ºC o quan i ica ion o in acellula i al con en .
2.5. Analy ical Me hods
Cell concen a ion and iabili y we e de e mined by coun ing cells on a
Fuchs-Rosen hal haemocy ome e (B and, We heim, Ge many) using he
ypan blue (In i ogen) dye exclusion me hod.
AV i a ion was pe o med by he end-poin dilu ion me hod (TCID50) using
96 well pla es and 293 cells. The i e was de e mined acco ding o he
me hod o Spea man and K abe , as desc ibed elsewhe e (Da ling e al.,
1998).
Glucose and lac a e we e analyzed using a YSI Mul ipa ame e Bioanaly ical
Sys em Model 7100 MBS (Yellow Sp ings, USA).
Ammonia was quan i ied enzyma ically using a UV es numbe 1112732035
(Boeh inge Mannheim, R-Biopha m AG, Ge many).
Chap e III.1
68
Comple e analysis o amino acid composi ion was done by HPLC using a
Pico-Tag (Wa e s) sys em. Amino acids we e de i a ized wi h he phenyl-
iso hiociana e me hod (Ma sudai a 1990). The Pico-Tag sys em consis ed o
a No a-Pak C18 column (WAT 011695), a Wa e s 510 pump, and au oma ic
injec o Wa e s 717 and a unable Abso bance De ec o 486. Da a
acquisi ion was pe o med on a PC using so wa e om Millenium.
3. RESULTS AND DISCUSSION
3.1. E ec o CCI on AdV p oduc ion
The e ec o CCI on he inal AdV p oduc ion yields was e alua ed in 2 L
bio eac o . In ec ion a CCI 1106 cell/ml (con ol), 2106 cell/ml and
3106 cell/ml wi h o wi hou comple e media exchange a he ime o
in ec ion was compa ed (Fig. 3.1.1). As can be obse ed, cell speci ic
p oduc i i y is no signi ican ly a ec ed a CCI 2106 cell/ml, when
compa ed wi h he con ol, leading o a 1.3 old inc ease on AdV
olume ic p oduc i i y. Howe e , one log dec ease on cell speci ic
p oduc i i y was obse ed a CCI 3106 cell/ml.
Medium exchange a he ime o in ec ion and he addi ion o glucose and
essen ial amino acids a 24 hpi has been epo ed o allow a sus ained
maximum speci ic p oduc i i y jus abo e 2106 cell/ml (Nadeau e al.,
1996). Figu e 3.1.1 shows ha a single medium exchange a he ime o
in ec ion is enough o main ain he cell speci ic p oduc i i y a he
maximum CCI o 3106 cell/ml, o which he highes olume ic
p oduc i i y was ob ained. The esul s ob ained a e imp o emen s on hose
epo ed in he li e a u e, sugges ing he se um and p o ein ee medium
used in his s udy (CD 293) o be a good choice o AdV p oduc ion a high
cell densi ies. Ne e heless, a p oduc ion scale, a medium exchange could
make he inal p oduc cos p ohibi i e. To de e mine how hese esul s
Re eed s a egies and non-ammoniagenic medium
69
a e ela ed o he me abolism o he cells, he kine ics o nu ien
consump ion and me aboli e p oduc ion we e e alua ed.
1E+01
1E+02
1E+03
1E+04
CCI 1 CCI 1+
media
exchange
CCI 2 CCI 2+
media
exchange
CCI 3 CCI 3 +
media
exchange
ip/cell
1E+08
1E+09
1E+10
1E+11
ip/ml
1E+01
1E+02
1E+03
1E+04
CCI 1 CCI 1+
media
exchange
CCI 2 CCI 2+
media
exchange
CCI 3 CCI 3 +
media
exchange
ip/cell
1E+08
1E+09
1E+10
1E+11
ip/ml
Figu e 3.1.1. E ec o media exchange and CCI in cell speci ic ( ) and AdV
olume ic ( ) p oduc i i ies. CCI 1, CCI 2 and CCI 3 co espond o 1, 2 and 3106
cell/ml a ime o in ec ion, espec i ely.
3.2. Cell me abolism e alua ion a di e en CCIs
Cell me abolism was assessed be o e and a e in ec ion a CCI 1106
cell/ml and 3106 cell/ml. A e in ec ion a CCI 1106 cell/ml, inc eases
o 1.3 old in glu amine and 1.8 old in glucose speci ic consump ion a es
we e obse ed, concomi an wi h inc eases o 1.2 old in ammonia and 3.9
old in lac a e speci ic p oduc ion a es (da a no shown). No signi ican
al e a ion in he mola a io o ammonia p oduced/glu amine consumed
was obse ed, whe eas he mola a io o lac a e p oduced/glucose
consumed inc eased 2.3 imes (Table 3.1.1). Elias e al. (2003) obse ed
ha o 293 cells, he lux o glucose in o he TCA cycle and i s subsequen
u iliza ion is limi ed as a esul o he lack o ce ain key enzymes in his
pa hway. This may be he explana ion o he signi ican inc ease in he
mola a io o lac a e p oduced/glucose consumed obse ed in ou
Chap e III.1
76
ip/cell
ip/ml
1E+08
1E+09
1E+10
1E+11
1E+01
1E+02
1E+03
1E+04
Gln CCI 1 Gln CCI 3 Glu CCI 1 Glu CCI 3
ip/cell
ip/ml
1E+08
1E+09
1E+10
1E+11
1E+01
1E+02
1E+03
1E+04
Gln CCI 1 Gln CCI 3 Glu CCI 1 Glu CCI 3
Figu e 3.1.7. E ec o medium supplemen in cell speci ic ( ) and AdV olume ic
() p oduc i i ies. Gln and Glu co espond o glu amine and glu ama e
supplemen ed medium and CCI 1 and CCI 3 co espond o 1 and 3106 cell/ml a
ime o in ec ion, espec i ely.
These esul s sugges an inhibi o y e ec on AdV p oduc ion by ammonia
accumula ion and con i med ha he use o non-ammoniagenic medium
imp o ed AdV olume ic p oduc i i y. Howe e , he ammonia
accumula ion seems no o be he only inhibi o y ac o on AdV p oduc ion,
since in op imal condi ion, a 3 imes inc ease on AdV olume ic
p oduc i i y was o be expec ed a CCI 3×106 cell/ml, when only an 1.8
old inc ease was ob ained, co esponding o he highes dec ease in cell
speci ic p oduc i i y indica ed abo e.
4. CONCLUSIONS
The p esen s udy shows ha by using CD 293 medium he AdV p oduc ion
eaches he highes olume ic p oduc i i y epo ed o an in ec ion
pe o med a CCI o 2106 cell/ml in ba ch mode. Mo eo e , AdV
p oduc i i y can be imp o ed e en a a CCI o 3106 cell/ml wi h a single

Re eed s a egies and non-ammoniagenic medium
77
CD 293 medium exchange a he ime o in ec ion. Ne e heless, o scaled
up p ocesses, a medium exchange s ep is no cos e ec i e.
Glu amine, h eonine and glucose we e ound o be limi ing nu ien s a
CCI o 3106 cell/ml, al hough no imp o emen on AdV p oduc i i y could
be achie ed a e he addi ion o hese nu ien s a he ime o in ec ion.
On he o he hand, ammonia was a an inhibi o y concen a ion o
in ec ion a his CCI. By adap ing he cells o non-ammoniagenic medium,
an 1.8 old inc ease in AdV olume ic p oduc i i y a CCI 3106 cell/ml
was ob ained, al hough he “ideal” si ua ion would ha e been an inc ease
o 3 imes a his CCI; hus, ammonia is an impo an pa ame e o be
conside ed o in ec ion a high cell densi ies, bu no he only.
Fu u e imp o emen s o he cul u e p ocess may come om analysis o
o he nu ien s han he usual glucose, lac a e, ammonia and amino acids.
A cock ail o di e en nu ien s a e p esen in he cul u e media ha may
ha e an impo an impac on AdV p oduc ion as i amins, lipids, ho mones
and g ow h ac o s (see Annex B). Combina ion o hese measu emen s may
lead o he design o a new s a egy pe mi ing a highe cell speci ic and,
consequen ly, AdV olume ic p oduc i i y.
5. ACKNOWLEDGEMENT
The au ho s a e g a e ul o D Tom Ba e (IAH-Pi b igh , UK) o p o iding
he ecombinan adeno i us and Eng Paula Chicau o p o iding da a om
he amino acids analysis se ice a he Ins i u o de Tecnologia Química e
Biológica, Uni e sidade No a de Lisboa, Oei as, Po ugal. The au ho s
acknowledge and app ecia e he inancial suppo ecei ed om he
Eu opean Commission (P ojec ICF599A4PR01) and om Fundação pa a a
Ciência e Tecnologia – Po ugal (P ojec POCTI/BIO/46515/2002 and
s uden g an SFRH/BD/10614/2002).
Chap e III.1
78
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Soma iba, S. and Bisch, D., 2000. An imp o ed anion-exchange HPLC me hod o
he de ec ion and pu i ica ion o adeno i al pa icles. Gene The 7, 1055-1062.
Ch is ie, A. and Bu le , M., 1999. The adap a ion o BHK cells o a non-
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kidney cell cul u e. Appl Mic obiol Bio echnol 51, 579-585.
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E ec s o ammonia and lac a e on g ow h, me abolism, and p oduc i i y o BHK
cells. Enzyme Mic ob Technol 27, 43-52.
Da ling, A.J., Boose, J.A. and Spal o, J., 1998. Vi us assay me hods: accu acy and
alida ion. Biologicals 26, 105-10.
Elias, C.B., Ca pen ie , E., Du oche , Y., Bisson, L., Wagne , R. and Kamen, A.,
2003. Imp o ing glucose and glu amine me abolism o human HEK 293 and
T ichoplusia ni insec cells enginee ed o exp ess a cy osolic py u a e ca boxylase
enzyme. Bio echnol P og 19, 90-97.
Eloi , M. and Adam, M., 1995. Isogenic adeno i uses ype 5 exp essing o no
exp essing he E1A gene: e iciency as i us ec o s in he accina ion o pe missi e
and non-pe missi e species. J Gen Vi ol 76, 1583-1589.
Ga nie , A., Co e, J., Nadeau, I., Kamen, A. and Massie, B., 1994. Scale-up o he
adeno i us exp ession sys em o he p oduc ion o ecombinan p o ein in human
293S cells. Cy o echnology 15, 145-155.
Re eed s a egies and non-ammoniagenic medium
79
Hen y, O., Do mond, E., Pe ie , M. and Kamen, A., 2004. Insigh s in o adeno i al
ec o p oduc ion kine ics in acous ic il e -based pe usion cul u es. Bio echnol
Bioeng 86, 765-774.
Huyghe, B.G., Liu, X., Su jip o, S., Suga man, B.J., Ho n, M.T., Shepa d, H.M.,
Scandella, C.J. and Shab am, P., 1995. Pu i ica ion o a ype 5 ecombinan
adeno i us encoding human p53 by column ch oma og aphy. Hum Gene The 6,
1403-16.
Iye , P., Os o e, J.M. and Vacan e, D., 1999. Compa ison o manu ac u ing
echniques o adeno i us p oduc ion. Cy o echnology 30, 169-172.
Ja don, M. and Ga nie , A., 2003. pH, pCO2, and Tempe a u e E ec on R-
Adeno i us P oduc ion. Bio echnol P og 19, 202-208.
Kamen, A. and Hen y, O., 2004. De elopmen and op imiza ion o an adeno i us
p oduc ion p ocess. J Gene Med 6 Suppl 1, S184-192.
Ma sudai a, P. 1990. Me hods in enzymology. P ess, A., edi o . San Diego.
McQueen, A. and Bailey, J.E., 1991. G ow h inhibi ion o hyb idoma cells by
ammonium ion: co ela ion wi h e ec s on in acellula pH. Biop Eng 6, 49-61.
Moun ain, A., 2000. Gene he apy: he i s decade. T ends Bio echnol 18, 119-128.
Nadeau, I., Ga nie , A., Cô é, J., Massie, B., Cha a ie, C. and Kamen, A., 1996.
Imp o emen o ecombinan p o ein p oduc ion wi h human adeno i us/293S
exp ession sys em using ed-ba ch S a egies. Bio echnol Bioeng 51, 613-623.
Nadeau, I., Gilbe , P.A., Jacob, D., Pe ie , M. and Kamen, A., 2002. Low-p o ein
medium a ec s he 293SF cen al me abolism du ing g ow h and in ec ion wi h
adeno i us. Bio echnol Bioeng 77, 91-104.
Nadeau, I., Jacob, D., Pe ie , M. and Kamen, A., 2000a. 293SF Me abolic Flux
analysis du ing cell g ow h and in ec ion wi h an adeno i al ec o . Bio echnol P og
16, 872-884.
Chap e III.1
80
Nadeau, I. and Kamen, A., 2003. P oduc ion o adeno i us ec o o gene he apy.
Bio echnol Ad 20, 475-489.
Nadeau, I., Saba ie, J., Koehl, M., Pe ie , M. and Kamen, A., 2000b. Human 293
cell me abolism in low glu amine-supplied cul u e: in e p e a ion o me abolic
changes h ough me abolic lux analysis. Me ab Eng 2, 277-292.
Rea, D., Schagen, F.H., Hoeben, R.C., Meh ali, M., Ha enga, M.J., Toes, R.E.,
Melie , C.J. and O inga, R., 1999. Adeno i uses ac i a e human dend i ic cells
wi hou pola iza ion owa d a T-helpe ype 1-inducing subse . J Vi ol 73, 10245-
10253.
Xie, L., Pilb ough, W., Me allo, C., Zhong, T., Pikus, L., Leung, J., Aunins, J.G.
and Zhou, W., 2002. Se um- ee suspension cul i a ion o PER.C6R cells and
ecombinan adeno i us p oduc ion unde di e en pH condi ions. Bio echnol
Bioeng 80, 569-579.
Ammonia e ec on pHi and AdV p oduc ion
81
PART 2
E ec o ammonia p oduc ion on in acellula pH: consequen e ec on
adeno i us ec o p oduc ion
Fe ei a T.B., Ca ondo M.J.T., Al es P.M. (2007) J. Bio echnol., 119:272-
280.

Chap e III.2
82
Abs ac
Recombinan adeno i al ec o s (AdV) ha e p o en o be highly e icien
o he deli e y and exp ession o o eign genes in a b oad spec um o cell
ypes and species bo h o accina ion and gene he apy in a numbe o
speci ic applica ions.
In his s udy, he e ec o ammonia p oduc ion on in acellula pH (pHi)
and consequen ly inhibi ion o AdV p oduc ion a high cell densi ies is
assessed. Di e en speci ic ammonia p oduc ion a es we e ob ained o
293 cells adap ed o g ow in glu ama e supplemen ed medium (non-
ammoniagenic medium) as compa ed wi h 293 cells g owing in glu amine
supplemen ed medium (ammoniagenic medium); pHi was obse ed o be
lowe du ing cell g ow h and AdV p oduc ion a bo h high and low CCI in
he ammoniagenic medium, whe e he speci ic ammonia p oduc ion a e is
highe . In addi ion, a e in ec ion a CCI o 3106 cell/ml, he cell iabili y
dec eased signi ican ly in he ammoniagenic medium, a ibu ed o he
ac i a ion o an acidic pa hway o apop osis. Fu he mo e, AdV DNA was
obse ed o be deg aded a he obse ed pHi in he ammoniagenic medium,
dec easing signi ican ly he amoun o AdV DNA a ailable o
encapsida ion. To elucida e he pHi e ec upon AdV p oduc ion, 293 cells
we e in ec ed a a CCI o 1106 cell/ml in he non-ammoniagenic medium
wi h a manipula ed pHi as obse ed a he ime o in ec ion a CCI o 3106
cell/ml in he ammoniagenic (pHi 7.0) and non-ammoniagenic (pHi 7.3)
media; AdV olume ic p oduc i i ies we e obse ed o be lowe when he
cells we e exposed o he lowe pHi. Thus, he impo ance o con olling all
he ac o s con ibu ing o pHi on AdV p oduc ion, such as ammonia
p oduc ion, has been es ablished.
Ammonia e ec on pHi and AdV p oduc ion
83
CONTENTS
1. INTRODUCTION_________________________________________ 84
2. MATERIALS AND METHODS ________________________________ 86
2.1. Cell line and medium ________________________________ 86
2.2. In ec ion in shake lasks ______________________________ 87
2.3. Vi us cul u e samples p epa a ion ______________________ 87
2.4. Adeno i al ec o i a ion ____________________________ 88
2.5. In acellula pH measu emen _________________________ 88
2.6. DNase II ac i i y assay a di e en pH alues______________ 89
2.7. De e mina ion o adeno i us DNA by quan i a i e eal ime PCR
_____________________________________________________ 89
2.8. Analy ical Me hods __________________________________ 90
3. RESULTS AND DISCUSSION ________________________________ 90
3.1. E ec o ammonia addi ion on adeno i us ec o p oduc ion _ 90
3.2. E ec o ammonia on in acellula pH du ing cell g ow h ____ 91
3.3. E alua ion o ammonia p oduc ion and in acellula pH du ing
adeno i us ec o p oduc ion a di e en CCIs ________________ 93
3.4. DNase II e ec upon adeno i us ec o DNA a di e en pHs__ 96
3.5. E ec o in acellula pH on adeno i us ec o p oduc ion ___ 97
4. CONCLUSIONS__________________________________________ 98
5. ACKNOWLEDGEMENT ____________________________________ 98
6. REFERENCES__________________________________________ 100
Chap e III.2
84
1. INTRODUCTION
Recombinan adeno i al ec o s (AdV) ha e p o en o be highly e icien
o he deli e y and exp ession o o eign genes in a b oad spec um o cell
ypes and species, in a numbe o speci ic applica ions (Volpe s and
Kochanek, 2004; Fe ei a e al., 2005a). Thus, as he ma ke needs o AdV
a e inc easing, me hodologies o p oduc ion o concen a ed AdV a e
needed. Al hough 293 cells can be g own up o 8106 cell/ml in ba ch
sys ems (Fe ei a e al., 2005c), AdV p oduc ion a high cell densi ies is
limi ed by he so-called “cell densi y e ec ”, i.e., a d op in cell speci ic
p oduc i i y concomi an wi h inc eased cell concen a ion a in ec ion
(CCI) o alues abo e 1106 cells/ml (Nadeau and Kamen, 2003; Kamen
and Hen y, 2004; Fe ei a e al., 2005b). Al hough his e ec has been
a ibu ed mainly o nu ien limi a ions and/o byp oduc s accumula ion,
he exac na u e o he ac o s limi ing AdV p oduc i i ies a high CCIs
emains unknown. P e ious s udies ha e indica ed ha medium
eplacemen a he ime o in ec ion and he addi ion o glucose and amino
acids a 24 h pos in ec ion (hpi) oge he wi h pe iodic pH adjus men s,
allows o high cell speci ic p oduc i i y a cell densi ies in he ange 2106
o 3106 cells/ml (Ga nie e al., 1994; Nadeau e al., 1996). Ou g oup has
shown ha ammonia is an impo an pa ame e o in ec ion a high cell
densi ies, since, by adap ing 293 cells o non-ammoniagenic medium, a 5
old inc ease on cell speci ic p oduc i i y could be ob ained a a CCI o
3×106 cell/ml (Fe ei a e al., 2005b). Howe e , he mechanisms by which
ammonia inhibi s AdV p oduc ion emain unknown.
Ammonia de i es mainly om deamina ion o glu amine, whose
concen a ion in he cul u e medium is usually 5 o 20 old highe han ha
o o he amino acids (Eagle, 1955). Comple e ca abolism o glu amine
p oduces 2 mol o ammonia pe mol o glu amine. O he mino sou ces o
ammonia include he spon aneous deg ada ion o glu amine (Oz u k and
Palsson, 1990; A ii e al., 1999) and ca abolism o o he amino acids.
Ammonia e ec on pHi and AdV p oduc ion
85
Se e al mechanisms o ammonia oxici y ha e been desc ibed in animal
cells, including: dis u bance o elec ochemical g adien s (Mi abe e al.,
1997); inhibi ion o enzyme eac ions; changes in in acellula pH (pHi)
(McQueen and Bailey, 1990); inc eased demand o ene gy main enance
(Ma inelle and Haggs om, 1993); inhibi ion o cell g ow h; and pe u bed
p ocessing and sec e ion o p o eins and apop osis induc ion (C uz e al.,
2000).
H+
-
pH H+
H+
+NH4+H+NH3
+
NH4+H+NH3
+
-
Na+
Na+
osmolali y
+Na+
+NH4+H+NH3
+
NH4+H+NH3
+
Cy oplasm
Cul u e Medium
Cell
memb ane
H+
-
pH H+
H+
H+
H+
+NH4+H+NH3
+
NH4+H+NH3
+
NH4+H+NH3
+
NH4+H+NH3
+
-
Na+
Na+
osmolali y
+Na+
+NH4+H+NH3
+
NH4+H+NH3
+
NH4+H+NH3
+
NH4+H+NH3
+
Cy oplasm
Cul u e Medium
Cell
memb ane
Figu e 3.2.1 Mechanisms in luencing pHi a dec eased medium pH, inc eased
ammonium ion concen a ion, and inc eased medium osmolali y (adap ed om
Che le and Ma c (1998)).
In he case o pHi, ammonia can dis u b he op imal cy oplasma ic pH by
o ming a “p o on shu le” ( ep esen ed schema ically in Figu e 3.2.1):
b ie ly, he in acellula ly o igina ed ammonium ions (NH4+) a e apidly
exc e ed by passi e di usion, unde he o m o ammonia ions (NH3), o
he ex acellula milieu wi h concomi an accumula ion inside he cell o
one p o on (H+) pe NH3 molecule eleased. This exc e ion is acili a ed by
he nega i e cha ge o he memb ane po en ial and he g adien o
ammonia ions ac oss he cell memb ane. Once ou side he cell, NH4+can
en e he cell again p opelled by he la ge elec ical g adien due o he
highly cha ged memb ane and lea e he cell again unde he NH3 o m; his
cycle can be s a ed o e and o e again (McQueen and Bailey, 1990; Wu e
Chap e III.2
92
medium (qammonia = 410-9 mmol/cell.h) in e ms o g ow h, ammonia
p oduc ion and pHi. As shown in Figu e 3.2.3, o he non-ammoniagenic
medium, he cells we e able o main ain he pHi be ween 7.2 and 7.3
h oughou all he cul u e; o ammoniagenic medium he pHi was
conside ably lowe : a d op o 0.2 uni s was e iden a ime 0 h and, as cells
eached he concen a ion o 2106 cell/ml (a 96 h), a signi ican dec ease
in pHi was egis e ed.
Fo he non-ammoniagenic medium, he pHe was main ained a 7.20 du ing
all he cul u e ime. Fo he ammoniagenic medium, a dec ease o 0.05
( om 7.20 o 7.15) was obse ed om ime 0 o ime 96; howe e , his
dec ease was no as signi ican as he one obse ed o he pHi.
0E+00
1E+06
2E+06
3E+06
4E+06
5E+06
6E+06
0 24 48 72 96 120 144 168 192 216 240
ime (h)
Viable cells (cell/ml)
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
pHi
0E+00
1E+06
2E+06
3E+06
4E+06
5E+06
6E+06
0 24 48 72 96 120 144 168 192 216 240
ime (h)
Viable cells (cell/ml)
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
pHi
Figu e 3.2.3. E alua ion o pHi (emp y symbols) du ing 293 cells g ow h ( ull
symbols) in ammoniagenic medium (ci cles) and non-ammoniagenic medium
( iangles).
By moni o ing ammonia concen a ion i is possible o co ela e he
changes obse ed in pHi wi h he ammonia p oduc ion (Figu e 3.2.4). As
expec ed, o he non-amoniagenic medium he p oduc ion o ammonia

Ammonia e ec on pHi and AdV p oduc ion
93
was signi ican ly lowe han o he ammoniagenic medium. The obse ed
delay in pHi dec ease ela i e o he as e enhancemen o ammonia
concen a ion in he cul u e medium can be ela ed wi h he inc easing
numbe o “p o on shu le” cycles (McQueen and Bailey, 1990; Wu e al.,
1993) leading o a much highe up ake o p o ons hen he o e all cell
capaci y o exc e e hem.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0 24 48 72 96 120 144 168 192 216 240
ime (h)
Ammonia (mM)
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
pHi
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0 24 48 72 96 120 144 168 192 216 240
ime (h)
Ammonia (mM)
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
pHi
Figu e 3.2.4. Rela ionship be ween pHi (emp y symbols) and ammonia p oduc ion
( ull symbols) in ammoniagenic medium (ci cles) and non-ammoniagenic medium
( iangles).
3.3. E alua ion o ammonia p oduc ion and in acellula pH du ing
adeno i us ec o p oduc ion a di e en CCIs
In o de o e alua e he e ec o p oduced ammonia on pHi du ing he
p oduc ion o AdV a a CCI o 1 and 3×106 cell/ml, 293 cells we e in ec ed
in ei he non-ammoniagenic and ammoniagenic media. As can be obse ed
in Figu e 3.2.5, he pHi is se e ely a ec ed a e in ec ion, independen ly
o he medium and CCI used. Howe e , o bo h cul u e media es ed, his
Chap e III.2
94
dec ease is mo e p onounced when he in ec ion is pe o med a CCI o
3×106 cell/ml, especially o ammoniagenic media.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0 6 12 18 24 30 36 42 48
ime (hpi)
Ammonia (mM)
5.7
5.8
5.9
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
pHi
CCI = 1×106cell/ml
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0 6 12 18 24 30 36 42 48
ime (hpi)
Ammonia (mM)
5.7
5.8
5.9
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
pHi
CCI = 1×106cell/ml
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0 6 12 18 24 30 36 42 48
ime (hpi)
Ammonia (mM)
5.7
5.8
5.9
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
pHi
CCI = 3×106cell/ml
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
0 6 12 18 24 30 36 42 48
ime (hpi)
Ammonia (mM)
5.7
5.8
5.9
6.0
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
7.0
7.1
7.2
7.3
pHi
CCI = 3×106cell/ml
Figu e 3.2.5. E alua ion o pHi (emp y symbols) and ammonia ( ull symbols) du ing
AdV p oduc ions a CCI o 1106 cell/ml in ammoniagenic medium (squa es) and
non-ammoniagenic medium (diamonds) and CCI o 3106 cell/ml in ammoniagenic
medium (ci cles) and non-ammoniagenic medium ( iangles) wi h a MOI o 10.
Ammonia e ec on pHi and AdV p oduc ion
95
The speci ic ammonia p oduc ion a e was no signi ican ly a ec ed when
he CCI inc eased om 1 o 3×106 cell/ml, hus he obse ed pHi dec ease
(Figu e 3.2.5) seems o be p omo ed by o he ac o s han he ammonia
p oduc ion. Ma suyama e al. (2000) shown ha a e y ea ly e en in
apop osis in ol es a change on he pHi egula ion, meaning ha he
signi ican dec ease obse ed in pHi a e AdV in ec ion may be a way o
he cell espond o in ec ion by en e ing in apop osis. In addi ion,
Ma suyama e al. (2000) ha e shown ha he e iciency o caspase
ac i a ion, esponsible o he beginning o he apop osis p ocess, is pH
sensi i e, wi h an in i o pH op imum o app oxima ely 6.6. In ac , he
amoun o iable cells a e in ec ion a CCI o 3106 cell/ml s a o
unde go a mo e p onounced dec ease when he pHi eaches he alue o
6.6 (Figu e 3.2.5) a 24 hpi o he ammoniagenic medium e sus 36 hpi o
he non-ammoniagenic medium (Figu e 3.2.6); his means ha cells a e
kep iable a e in ec ion o a longe ime when he non-ammoniagenic
medium is used.
0.0E+00
5.0E+05
1.0E+06
1.5E+06
2.0E+06
2.5E+06
3.0E+06
3.5E+06
0 6 12 18 24 30 36 42 48
ime (hpi)
Viable cells (cell/ml)
0
5
10
15
20
25
30
% dead cells
0.0E+00
5.0E+05
1.0E+06
1.5E+06
2.0E+06
2.5E+06
3.0E+06
3.5E+06
0 6 12 18 24 30 36 42 48
ime (hpi)
Viable cells (cell/ml)
0
5
10
15
20
25
30
% dead cells
Figu e 3.2.6. E ec o CCI o 3106 cell/ml in cell iabili y ( ull symbols) and
pe cen age o dead cells (emp y symbols) in ammoniagenic medium (ci cles) and
Chap e III.2
96
non-ammoniagenic medium ( iangles).
3.4. DNase II e ec upon adeno i us ec o DNA a di e en pHs
To e alua e how pHi a ec s DNase II ac i i y and consequen ly AdV DNA,
incuba ions o he i al DNA we e done a di e en pH alues. As shown in
Figu e 3.2.7, pH a ec s he ac i i y o DNase II upon he AdV DNA, a
signi ican decline being obse ed o pH alues below 6.6. No signi ican
e ec s in AdV DNA we e obse ed o simila s udies pe o med wi h DNase
I (da a no shown).
1E+03
1E+04
1E+05
1E+06
1E+07
I0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6
pH
DNA (copies/ml)
1E+03
1E+04
1E+05
1E+06
1E+07
I0 7.8 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6
pH
DNA (copies/ml)
Figu e 3.2.7. E ec o pH on he ac i i y o DNase II. AdV DNA was incuba ed wi h
a cell supe na an ex ac as desc ibed in ma e ial and me hods. () Rep esen s
he ini ial amoun o AdV DNA obse ed a ime 0 o each pH es ed.
Compa ing he ac i i y o DNase II wi h he pHi p o iles a e in ec ion a a
CCI o 1 and 3106 cell/ml in he ammoniagenic and non-ammoniagenic
medium (Figu e 3.2.5), i is possible o conclude ha a CCI o 1106
cell/ml, independen ly o he medium used, only a he end o he
p oduc ion p ocess he pHi each alues below 6.6. On he o he hand,
when cells a e in ec ed a CCI o 3106 cell/ml in he ammoniagenic
medium, he pHi each he 6.6 uni s as e han in he non-ammoniagenic
Ammonia e ec on pHi and AdV p oduc ion
97
medium. Thus, o ammoniagenic media, due o he signi ican inc ease on
DNase II ac i i y o alues below pH 6.6, mo e AdV DNA deg ada ion will
occu leading o less a ailabili y o i us DNA o encapsida ion wi h a
consequen dec ease in bioac i e AdV p oduc ion.
3.5. E ec o in acellula pH on adeno i us ec o p oduc ion
To elucida e he pHi e ec upon AdV p oduc ion, 293 cells adap ed o g ow
in he non-ammoniagenic medium we e in ec ed a a CCI o 1106 cell/ml
a pH 7.0 o a pH 7.3 (pHi obse ed a he ime o in ec ion a CCI o 3106
cell/ml in he ammoniagenic and non-ammoniagenic media espec i ely),
wi h o wi hou nige icin addi ion and ha es ed a 48 hpi. Nige icin was
used o mimic he pHi obse ed a CCI 3106 cell/ml in bo h media a pHi
7.0 and 7.3.
1E+07
1E+08
1E+09
1E+10
pH 7.0
(con ol)
pH 7.3
(con ol)
pH 7.0 pH 7.3
ip/ml
1E+07
1E+08
1E+09
1E+10
(con ol) (con ol)
ip/ml
1E+07
1E+08
1E+09
1E+10
pH 7.0
(con ol)
pH 7.3
(con ol)
pH 7.0 pH 7.3
ip/ml
1E+07
1E+08
1E+09
1E+10
(con ol) (con ol)
ip/ml
Figu e 3.2.8. E ec o pHi on AdV olume ic p oduc i i y. pHi was dec eased by
addi ion o 30 mM KCl and o 50 M nige icin as desc ibed in ma e ial and me hods.
As can be obse ed in Figu e 3.2.8, al hough he e is no signi ican
di e ence on AdV p oduc ion a pH 7.0 o a pH 7.3 in con ol condi ions,

Chap e III.2
98
when nige icin is used a d op in AdV olume ic p oduc i i y is obse ed o
bo h pHs, al hough i is mo e p onounced a pH 7.0. Despi e he ac ha
nige icin has a esidual cy o oxic e ec (da a no shown), i is ob ious
ha , o a pH o 7.0, AdV olume ic p oduc i i y is s ongly a ec ed.
These esul s e lec he impac o he pHi on AdV p oduc ion and he
o e all need o minimize all he ac o s ha con ibu e o i s dec ease,
such as he in acellula ammonia p oduc ion.
4. CONCLUSIONS
The p esen s udy compa es he e ec o wo di e en cul u e media
leading o di e en speci ic ammonia p oduc ion a es, upon pHi and AdV
p oduc i i y in 293 cells in ec ed a di e en CCIs (1 and 3×106 cell/ml).
Du ing cell g ow h he pHi dec eased signi ican ly in he ammoniagenic
medium, whe e he speci ic ammonia p oduc ion a e is highe ;
u he mo e, a e in ec ion, he pHi d opped signi ican ly a bo h CCIs o
bo h media, al hough he lowes pHi was ob ained o he ammoniagenic
medium. These changes obse ed in he pHi we e ound o play an
impo an ole on bo h p ocesses o apop osis and AdV DNA deg ada ion,
leading o a dec ease in bioac i e AdV p oduc ion, mo e se e e o he
ammoniagenic medium.
The da a epo ed he ein iden i ies pHi as one o he key ac o s
esponsible o he “cell densi y e ec ” obse ed on AdV p oduc ion. Non-
ammoniagenic medium was shown as a good op ion o be implemen ed a
he bio eac ion le el ha will lead o he main enance o cell iabili y
du ing AdV p oduc ion and consequen inc ease on cell speci ic
p oduc i i y.
5. ACKNOWLEDGEMENT
The au ho s a e g a e ul o D Tom Ba e (Ins i u e o Animal Heal h-
Ammonia e ec on pHi and AdV p oduc ion
99
Pi b igh , UK) o p o iding he ecombinan adeno i us, Eng Ma lene
Ca mo o all he suppo conce ning he low cy ome ic analyzes and Eng
Rosá io Clemen e o PCR analysis. The au ho s acknowledge and
app ecia e he inancial suppo ecei ed om he Eu opean Commission
(P ojec RP/PPR ORALVAC ICA4-CT-2000-30027) and om Fundação pa a a
Ciência e Tecnologia, Po ugal (P ojec POCTI/BIO/46515/2002 and s uden
g an SFRH/BD/10614/2002).
Chap e III.2
100
6. REFERENCES
A ii, K., Kobayashi, H., Kai, T., and Kokuba, Y., 1999. Deg ada ion kine ics o L-
glu amine in aqueous solu ion. Eu J Pha m Sci 9, 75-8.
Ba y, M. A., and Eas man, A., 1993. Iden i ica ion o deoxy ibonuclease II as an
endonuclease in ol ed in apop osis. A ch Biochem Biophys 300, 440-50.
Che le , M., and Ma c, A., 1998. In acellula pH moni o ing as a ool o he s udy
o hyb idoma cell beha io in ba ch and con inuous bio eac o cul u es. Bio echnol
P og 14, 626-38.
C uz, H. J., F ei as, C. M., Al es, P. M., Mo ei a, J. L., and Ca ondo, M. J., 2000.
E ec s o ammonia and lac a e on g ow h, me abolism, and p oduc i i y o BHK
cells. Enzyme Mic ob Technol 27, 43-52.
Eagle, H., 1955. Nu i ion needs o mammalian cells in issue cul u e. Science 122,
501-14.
E ans, R. K., Naw ocki, D. K., Isopi, L. A., Williams, D. M., Casimi o, D. R., Chin,
S., Chen, M., Zhu, D. M., Shi e , J. W., and Volkin, D. B., 2004. De elopmen o
s able liquid o mula ions o adeno i us-based accines. J Pha m Sci 93, 2458-75.
Fe ei a, T. B., Al es, P. M., Aunins, J. G., and Ca ondo, M. J. T., 2005a. Use o
adeno i al ec o s as e e ina y accines. Gene The 12 Suppl 1, S73-83.
Fe ei a, T. B., Fe ei a, A. L., Ca ondo, M. J. T., and Al es, P. M., 2005b. E ec
o e eed s a egies and non-ammoniagenic medium on adeno i us p oduc ion a
high cell densi ies. J Bio echnol 119, 272-80.
Fe ei a, T. B., Fe ei a, A. L., Ca ondo, M. J. T., and Al es, P. M., 2005c. Two
di e en se um- ee media and osmolali y e ec upon 293 cell g ow h and
adeno i us p oduc ion. Bio echnol Le 27, 1809-13.
F elin, C., Vigne, P., Ladoux, A., and Lazdunski, M., 1988. The egula ion o he
in acellula pH in cells om e eb a es. Eu J Biochem 174, 3-14.
Ammonia e ec on pHi and AdV p oduc ion
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Ga nie , A., Co e, J., Nadeau, I., Kamen, A., and Massie, B., 1994. Scale-up o he
adeno i us exp ession sys em o he p oduc ion o ecombinan p o ein in human
293S cells. Cy o echnology 15, 145-155.
Go lieb, R. A., No dbe g, J., Skow onski, E., and Babio , B. M., 1996. Apop osis
induced in Ju ka cells by se e al agen s is p eceded by in acellula acidi ica ion.
P oc Na l Acad Sci U S A 93, 654-8.
Kamen, A., and Hen y, O., 2004. De elopmen and op imiza ion o an adeno i us
p oduc ion p ocess. J Gene Med 6 Suppl 1, S184-192.
Lagadic-Gossmann, D., Huc, L., and Lecu eu , V., 2004. Al e a ions o in acellula
pH homeos asis in apop osis: o igins and oles. Cell Dea h Di e 11, 953-61.
Ma inelle, K., and Haggs om, L., 1993. Mechanisms o ammonia and ammonium
ion oxici y in animal cells: anspo ac oss cell memb anes. J Bio echnol 30, 339-
50.
Ma suyama, S., Llopis, J., De e aux, Q. L., Tsien, R. Y., and Reed, J. C., 2000.
Changes in in ami ochond ial and cy osolic pH: ea ly e en s ha modula e caspase
ac i a ion du ing apop osis. Na Cell Biol 2, 318-25.
McQueen, A., and Bailey, J. E., 1990. E ec o Ammonium Ion and Ex acellula Ph
on Hyb idoma Cell-Me abolism and An ibody-P oduc ion. Bio echnol Bioeng 35,
1067-1077.
Mi abe , M., Na a o, A., Lopez, A., Canela, E. I., Mallol, J., Lluis, C., and F anco,
R., 1997. Ammonium oxici y in di e en cell lines. Bio echnology and
Bioenginee ing 56, 530-537.
Nadeau, I., Ga nie , A., Cô é, J., Massie, B., Cha a ie, C., and Kamen, A., 1996.
Imp o emen o ecombinan p o ein p oduc ion wi h human adeno i us/293S
exp ession sys em using ed-ba ch S a egies. Bio echnol Bioeng 51, 613-623.
Nadeau, I., and Kamen, A., 2003. P oduc ion o adeno i us ec o o gene
he apy. Bio echnol Ad 20, 475-489.
Chap e IV.1
108
1. INTRODUCTION
AdV ha e been ex ensi ely used as ec o s o bo h gene he apy and
ecombinan DNA accines, which ha e been es ed in bo h humans and
di e en animal species (Fe ei a e al., 2005a). The need o la ge
quan i ies o clinical-g ade AdV is an impo an limi a ion o in i o
expe imen a ions as well as o p e-clinical and clinical s udies (Nadeau and
Kamen 2003; Kamen and Hen y 2004). This limi a ion is due o he ac
ha he cell speci ic p oduc i i y dec eases sha ply wi h inc eased CCI
abo e 1106 cell/ml, he so called “cell densi y e ec ”. So a , his “cell
densi y e ec ” has been a ibu ed o nu ien limi a ions and/o
accumula ion o byp oduc s (Nadeau and Kamen 2003; Hen y e al., 2004;
Kamen and Hen y 2004; Fe ei a e al., 2005b). Al hough me abolic lux
analysis has been used o cha ac e ize he e ec o a ious en i onmen al
condi ions upon cell me abolism, he exac na u e o he ac o s limi ing
AdV p oduc i i ies a high cell densi y emains unknown (Nadeau e al.,
2000a; Nadeau e al., 2000b; Nadeau e al., 2002). The e o e, p esen
s a egies o o e coming his bo leneck ha e been ocused on he
imp o emen o cell g ow h en i onmen du ing and a e in ec ion. These
s a egies include medium eplacemen a he ime o in ec ion and he
addi ion o glucose a 24 hou pos in ec ion (hpi) oge he wi h pe iodical
pH adjus men s, allowing a sus ained maximum speci ic p oduc i i y a
1.6106 cell/ml (Ga nie e al., 1994). This s a egy, associa ed wi h
essen ial amino acids eeding a 24 hpi, is able o s abilise he olume ic
p oduc i i y a cell densi ies up o 2106 cell/ml (Nadeau e al., 1996).
Pe usion mode ope a ion has been demons a ed as he mos e ec i e
measu e o con ol he cul u e en i onmen and o emo e oxic
byp oduc s (Co in e al., 2004; Hen y e al., 2004; Kamen and Hen y
2004). Ne e heless, he cell speci ic p oduc i i y could only be main ained
by in ec ing cells a densi ies up o 3106 cell/ml using high pe usion a e
a 2 eac o olumes pe day (VVD), a 2 days pos -in ec ion, a e y cos ly

Impo ance o cell cycle
109
p oposi ion (Hen y e al., 2004; Kamen and Hen y 2004). By dec easing he
empe a u e o 35 oC pos in ec ion, Co in e al. (2004) ha e u he
inc eased CCI up o 8106 cell/ml a pe usion a e o 1 VVD wi h a
p oduc ion o 7.8109 in ec ious pa icles/ml (ip/ml). In s udies conduc ed
by he au ho s unde much less expensi e ed ba ch mode using 293 cells
adap ed o non-ammoniagenic medium an 1.8 old inc ease on AdV
olume ic p oduc i i y a CCI 3106 cell/ml was ob ained, when compa ing
wi h in ec ion a CCI 1106 cell/ml in he same medium, wi h a p oduc ion
o 8.5109 ip/ml (Fe ei a e al., 2005b; Fe ei a e al., 2005c).
Du ing cell cul u e p ocesses, especially in ba ch and ed-ba ch mode,
apa om he con inuously changing cul u e chemical and physical
en i onmen , he cells hemsel es a e also con inuously a ying hei
physiological and me abolic s a es. I is well known ha du ing he
cul u ing, he popula ion o cells goes h ough ou g ow h s ages, i.e., lag,
exponen ial, s a iona y and dea h-phase, whe e an indi idual cell commi s
o ou successi e g ow h phases in he cell cycle, i.e., G1 (gap one phase,
be ween mi osis and DNA syn hesis), S (DNA syn hesis phase), G2 (gap wo
phase, be ween comple ion o DNA syn hesis and mi osis) and M-phase
(mi osis phase). In some cases, cells may escape om he cell cycle and
en e a es ing phase (G0), om whe e, gi en sui able condi ion, hey can
e u n o he cell cycle. Du ing he cul u e p ocess, he cell cycle phase
dis ibu ion may be con inuously changing and no ma e in which phase o
he cell cycle he cells a e in ec ed by AdV, he i al DNA syn hesis will
only ake place 9h a e in ec ion (Hodge and Scha 1969).
The e ec o cell cycle phase a he ime o in ec ion on AdV p oduc i i y
has no been well desc ibed. The sca ce epo ed da a sugges s ha i is
bo h cell line and i us ype dependen . Fo a ian AdV, bo h p oduc ion o
in ec ious i us and i al DNA syn hesis a e co ela ed wi h e en s du ing
he S phase o he in ec ed chicken emb yo ib oblas s cells (K a and
Tische 1978). Fo HeLa cells, in ec ion wi h human Ad5 du ing S phase
p oduced g ea e yields o E1B 55-kDa-mu an and E4 o 6-mu an i uses
Chap e IV.1
110
han did cells in ec ed du ing G1 o asynch onous cells. Howe e he
p oduc ion o wild Ad and E4 o 3-mu an Ad was no signi ican ly
es ic ed by he cell cycle (Good um and O nelles 1997). In addi ion, o
many o he i uses, he p oduc ion is associa ed wi h he S phase o he
cell cycle. Fo example, coxsackie i us p oduc ion is dependen on G1 o
G1/S phase (Feue e al., 2002); o minu e i us o mice, a pa o i us, he
con e sion o he i al genome om inpu single-s anded DNA o a double-
s anded DNA, he o m which unde goes u he eplica ion, appea s o be
an S phase-speci ic e en (Wol e e al., 1980); in he baculo i us-insec
cell exp ession sys em, he in ec ion yield a G1 o S phase-in ec ion was
1.5-1.8- old highe han ha a G2/M phase-in ec ion (Sai o e al., 2002);
he eplica ion o he bo ine he pes i us-4 depends on ansi ion h ough S
phase (Vande plasschen e al., 1995); he he pes simplex i us ype 1
mw65 (VP16) inse ion mu an depends on cells in ec ed du ing S phase
o ea ly p o ein syn hesis and eplica ion (Daksis and P es on 1992).
Thus he aim o his wo k is o in es iga e he impo ance o he cell cycle
phase o he hos 293 cells a he ime o in ec ion upon AdV p oduc i i y,
so ha mo e e icien in ec ions a high cell densi y may be achie ed.
2. MATERIALS AND METHODS
2.1. Cell line and cul u e main enance
Suspension-adap ed 293 cells (ATCC-CRL-1573, Rock ille, MD) we e g own
in shake lasks (Co ning, NY) in comme cially a ailable and chemically
de ined medium wi hou se um and p o ein, CD293, supplemen ed wi h 4
mM o L-glu amine (all om, In i ogen, Glasgow, UK) a 160 pm unde an
humidi ied a mosphe e o 8% CO2 in ai a 37ºC. The cells we e ou inely
p opaga ed wice a week wi h an inoculum densi y o 0.5106cell/ml.
Impo ance o cell cycle
111
2.2. Cell in ec ion
A eplica ion-de ec i e AdV de i ed om ype 5 AdV was kindly p o ided by
D . Tom Ba e (IAH-Pi b igh , UK). In ec ion was ca ied ou in 125 ml
shake lasks wi h wo king olumes o 40 ml using a mul iplici y o in ec ion
(MOI) o 10 (see Annex A). In ec ed 293 cells we e ha es ed a 0, 24, 48
and 72 hpi and p epa ed as p e iously desc ibed in Fe ei a e al. (2005b)
2.3. Analy ical me hods
Cell concen a ion and iabili y we e de e mined by coun ing cells on a
Fuchs-Rosen hal haemocy ome e (B and, We heim, Ge many) using he
ypan blue (In i ogen, Glasgow, UK) dye exclusion me hod.
AdV i a ion was pe o med by he end-poin dilu ion me hod (TCID50)
using 96 well pla es and ancho age-dependen 293 cells cul u ed in T- lasks
(Sa s ed ); hese cells we e cul u ed in MEM supplemen ed wi h hea -
inac i a ed 5% e al bo ine se um (all om In i ogen, Glasgow, UK) and 2
mM L-glu amine unde an humidi ied a mosphe e o 5% CO2 in ai a 37 oC.
The i e was de e mined acco ding o he me hod o Spea man and
K abe , as desc ibed elsewhe e (Da ling e al., 1998).
Glucose and lac a e we e analyzed using an YSI Mul ipa ame e
Bioanaly ical Sys em Model 7100 MBS (Yellow Sp ings, USA).
Ammonia was quan i ied enzyma ically using he UV es numbe
1112732035 (Roche Diagnos ics GmbH, Mannheim, Ge many).
2.4. Flow cy ome ic analysis
To quan i y he cell-cycle dis ibu ion, cells we e s ained wi h p opidium
iodide o measu e he DNA con en by low-cy ome ic analysis (FACS).
Cells we e cen i uged a 200 g o 10 min a 4ºC, washed wi h ice-cold PBS
Chap e IV.1
112
and s ained wi h ice-cold Vindelo ’s solu ion (Vindelo e al., 1983): 1 g/l
isodic ci a e, pH 7.6 (Sigma); 50 mg/l p opidium iodide (Sigma); 0.1% NP
40 (Roche); 700 U/l RNase A (Sigma); 0.01 M NaCl (Me ck) o a inal cell
concen a ion o 1.5106cell/ml. All FACS analyses we e pe o med on a
FACSCalibu TM using he Cell Ques TM so wa e (Beck on-Dickinson, San
Jose, CA).
3. RESULTS AND DISCUSSION
3.1. Rela ionship be ween cell densi y and cell cycle phase
dis ibu ion du ing no mal ba ch cul u es o 293 cells
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6
Time a e inocula ion (days)
Viable cell densi y
0
10
20
30
40
50
60
70
Phase in he cell cycle
(pe cen age o popula ion)
0
1
2
3
4
5
6
7
8
0 2 3 4 5 6
0
10
20
30
40
50
60
70
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6
Viable cell densi yViable cell densi y
(106cell/ml)
0
10
20
30
40
50
60
70
Phase in he cell cycle
(pe cen age o popula ion)
Phase in he cell cycle
(pe cen age o popula ion)
0
1
2
3
4
5
6
7
8
0 2 3 4 5 6
0
10
20
30
40
50
60
70
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6
Time a e inocula ion (days)
Viable cell densi y
0
10
20
30
40
50
60
70
Phase in he cell cycle
(pe cen age o popula ion)
Phase in he cell cycle
(pe cen age o popula ion)
0
1
2
3
4
5
6
7
8
0 2 3 4 5 6
0
10
20
30
40
50
60
70
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6
Viable cell densi yViable cell densi y
(106cell/ml)
0
10
20
30
40
50
60
70
Phase in he cell cycle
(pe cen age o popula ion)
Phase in he cell cycle
(pe cen age o popula ion)
0
1
2
3
4
5
6
7
8
0 2 3 4 5 6
0
10
20
30
40
50
60
70
Figu e 4.1.1. P o iles o cell g ow h and cell cycle phase dis ibu ion du ing 293
cell suspension ba ch cul u es. Symbols: , cell densi y; , G2/M phase; , G1/G0
phase; ▲, S phase.
Figu e 4.1.1 desc ibes he cell cycle phase dis ibu ion a di e en cell
densi ies du ing a no mal ba ch cul i a ion p ocess o 293 cells. A e
inocula ion, he p opo ion o cells in he S phase o he cell cycle
Impo ance o cell cycle
113
inc eased sha ply, wi h a 70% inc ease wi hin he i s 24 h, om ca. 30%
o ca. 50% o he o al cell popula ion. Con e sely, he pe cen age o
G1/G0 cells in he cell cul u e ell du ing his pe iod. The highes S phase
p opo ion occu ed a he middle o he lag phase o he g ow h s age,
co esponding o a cell densi y o 0.6-0.8106cell/ml. The ea e , he
pe cen age o cells in he S phase dec eased g adually, as he cell densi y
inc eased abo e 1106cell/ml; co espondingly, he pe cen age o G1/G0
cells in he cell cul u e ose, while he pe cen age o cells a G2/M phase
was kep ela i ely s able a abou 11% du ing he whole g ow h cycle.
Accompanying he cell densi y inc ease, he nu ien s in he cul u e
medium, namely glucose and glu amine, dec eased quickly; a he same
ime, he accumula ion o he byp oduc s, specially lac a e and ammonia,
inc eased apidly in he 293 cell suspension ba ch cul u es (Figu e 4.1.2).
0
5
10
15
20
25
30
0 1 2 3 4 5 6
Time a e inocula ion (days)
Concen a ion o Glc and Lac (mM)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Concen a ion o Gln and NH
4
+(mM)
0
5
10
15
20
25
30
0 1 2 3 4 5 6
Time a e inocula ion (days)
Concen a ion o Glc and Lac (mM)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Concen a ion o Gln and NH
4
+(mM)
Figu e 4.1.2. Kine ics o o al glucose (Glc, ) and glu amine (Gln, )
consump ion and o al lac a e (Lac, ) and ammonia (NH4+, ∆) p oduc ion o 293
cell suspension ba ch cul u es.
Al hough Lullau e al. (2003) obse ed o 293-BACE-Fc a highe S phase

Chap e IV.1
114
ac ion a he end o ba ch cul u e, he end in he cell cycle phase
dis ibu ion o 293 cells ob ained he e is well suppo ed by o he s who
obse ed simila esul s o cul u es o hyb idoma cells (Rami ez and
Mu ha asan 1992; Balca cel and S ephanopoulos 2001; Luo and Yang 2004),
CHO cells (Ley and Tobey 1970; Tobey and Ley 1970) and Hela cells (Koza
and He bs 1992; Good um and O nelles 1997).
3.2. E ec o di e en p opo ion o cells unde S phase in equalized
cell densi y condi ions upon AdV p oduc i i y
As demons a ed abo e, he p opo ion o cells in he S phase dec eased as
he cell densi y ose. To e alua e i his dec eased p opo ion o cells in
he S phase a high cell densi y co ela es wi h he so called “cell densi y
e ec ”, cells we e in ec ed a di e en g ow h s ages wi h di e en
p opo ions a S phase unde equalized cell densi y condi ions a CCI o
1106 cell/ml. In b ie , a e ha es ing, he cells we e cen i uged o
o ally emo e he spen medium, and hen esuspended in esh medium
a a inal concen a ion o 1106 cell/ml be o e in ec ion, and in ec ed
wi h he same MOI a he same ime. As s a ed in he In oduc ion, he CCI
o 1106 cell/ml was chosen on he basis o he obse a ions ha i is he
op imum CCI o no mal AdV p oduc ion (Fe ei a e al., 2005c). The
medium exchange was pe o med o a oid lack o nu ien s and p esence
o oxic byp oduc hus pe mi ing o isola e he e alua ion o he cell
cycle e ec . The esul s a e illus a ed in Figu e 4.1.3 and summa ized in
Table 4.1.1.
Impo ance o cell cycle
115
1E+00
1E+01
1E+02
1E+03
1E+04
0 24 48 72
Time pos in ec ion (hou s)
ip/cell
0 24 48 72
1E+00
1E+01
1E+02
1E+03
1E+04
0 24 48 72
Time pos in ec ion (hou s)
ip/cell
0 24 48 72
1E+00
1E+01
1E+02
1E+03
1E+04
0 24 48 72
Time pos in ec ion (hou s)
ip/cell
0 24 48 72
1E+00
1E+01
1E+02
1E+03
1E+04
0 24 48 72
Time pos in ec ion (hou s)
ip/cell
0 24 48 72
Figu e 4.1.3. P o iles o cell speci ic p oduc i i y o cells ob ained om di e en
g ow h s ages wi h di e en p opo ions in S phase a he ime o in ec ion in
equalized cell densi y condi ions a a CCI o 1106 cell/ml. The 293 cells o his
expe imen we e ha es ed om lag, middle exponen ial, la e exponen ial and
s a iona y phases, co esponding o cell densi ies o 0.7, 1.6, 3.3, and 5106
cell/ml, wi h p opo ions o cells in S phase o 50% (▲), 40% (), 32% () and 28%
(Δ), espec i ely. The cells we e cen i uged o disca d he spen medium and
eplaced wi h esh medium a CCI o 1106 cell/ml, in o de o p o ide an
equalized in ec ion en i onmen al condi ion o all he cells.
Table 4.1.1. E ec o cells ob ained om di e en g ow h s ages wi h di e en
p opo ions in S phase a he ime o in ec ion in cell speci ic p oduc i i y a
he op imal cell concen a ion a in ec ion (1106 cell/ml). In ec ions we e
pe o med a MOI o 10 and AdV ha es ed a 72 hpi.
G ow h s age
o cells
O iginal cell
densi y (cell/ml)
Pe cen age o S phase
cells a in ec ion
Maximum cell speci ic
p oduc i i y (ip/cell)
Lag 0.710650%  3% 8.3103 0.7103
Middle
exponen ial 1.610640%  3% 4.7103 0.4103
La e
exponen ial 3.310632%  2% 2.5103 0.3103
S a iona y 5.0 28%  2% 1.3103 0.5103
Chap e IV.1
116
The in ec ion o cells om he lag phase, wi h he highes p opo ion a S
phase, esul ed in he highes p oduc i i y o 8.3103ip/cell a 72 hpi, 6.4
imes highe han he le el o cells ob ained om s a iona y phase wi h he
lowes p opo ion in S phase a he same ime (1.3103ip/cell). In o he
wo ds, e en hough all in ec ions we e pe o med a he op imal CCI (1106
cell/ml), cells ob ained om di e en g ow h s ages exhibi ed di e en
cell speci ic p oduc i i ies. These esul s s ongly suppo he hypo hesis
ha he cell speci ic p oduc i i y is dependen upon cell cycle a in ec ion,
i.e., highe p opo ion o cells a S phase, co esponding o a la ge cell
popula ion unde as e di ision, esul ed in highe AdV p oduc i i ies.
In e es ingly, cells ob ained om he g ow h s ages a highe cell densi ies
o 3.3 and 5.0106 cell/ml p esen ed a lowe cell speci ic p oduc ion a e
and a highe op imal ha es ing ime o 72 hpi al hough 48 hpi is he
op imal ha es ing ime o he cells ob ained om he g ow h s ages a
lowe cell densi ies o 0.7 and 1.6106 cell/ml (Figu e 4.1.3). Al hough his
e ec could be co ela ed wi h cell agg ega ion in he expe imen s
pe o med a high cell densi ies, no signi ican cell agg ega ion (no mo e
han 2-4 cells agg ega es) was ound. Mo eo e , an inc ease o 60% on
iable cell densi y a 24 hpi o cells wi h he highe p opo ion in S phase
was obse ed (Figu e 4.1.4). Hodge and Scha (1969) obse ed ha no
ma e in which phase o he cell cycle he cells a e in ec ed, he i al DNA
will s a o be p oduced a 9 hpi, meaning ha a cell in G1 phase will s op
a he S phase and ha a cell in he S phase will double once mo e a e
in ec ion; he e o e i is no su p ising o obse e cell g ow h a e
in ec ion whe e he p opo ion o cells on he S phase o he cell cycle is
highe .
Impo ance o cell cycle
117
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
0 24 48 72
Time pos in ec ion (hou s)
Viable cell densi y (106cell/ml)
0 24 48 720 24 48 72
Time pos in ec ion (hou s)
Viable cell densi y (106cell/ml)
0 24 48 72
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
0 24 48 72
Time pos in ec ion (hou s)
Viable cell densi y (106cell/ml)
0 24 48 720 24 48 72
Time pos in ec ion (hou s)
Viable cell densi y (106cell/ml)
0 24 48 72
Figu e 4.1.4. P o iles o cell g ow h a e in ec ion in equalized cell densi y
condi ions a a CCI o 1106 cell/ml. The 293 cells o his expe imen we e
ha es ed om lag, middle exponen ial, la e exponen ial and s a iona y phases,
co esponding o cell densi ies o 0.7, 1.6, 3.3, and 5106 cell/ml, wi h p opo ions
o cells in S phase o 50% (▲), 40% (), 32% () and 28% (Δ), espec i ely.
The e ec o di e en p opo ion o cells unde S phase a in ec ion on
AdV p oduc i i y was u he in es iga ed a highe CCI in he equalized
cell densi y condi ions. In his case, he CCI o 4106 cell/ml was chosen in
o de o highligh he di e ence in cell speci ic p oduc i i y in he
equalized cell densi y condi ion. As illus a ed in Figu e 4.1.5, simila ly o
he esul s achie ed a he op imal CCI o 1106 cell/ml, a he CCI o
4106 cell/ml he in ec ion o cell cul u es wi h highe p opo ion in S
phase also esul ed in highe cell speci ic p oduc i i y. A his CCI, he
in ec ion o cells wi h he highes S phase p opo ion (50%), esul ed in a
p oduc ion o 2.5103 ip/cell a 72 hpi. This cell speci ic p oduc i i y was
16.7- old highe han ha ob ained om in ec ion o cells om he
s a iona y g ow h s age a a cell densi y o 5106 cell/ml, consis ing o he
lowes S phase cell p opo ion (28%), and esul ed in he highe olume ic
p oduc i i y o 11010 ip/ml (Figu e 4.1.5). Once mo e, his cons i u e
s ong e idence ha he p opo ion o cells in S phase a in ec ion is an

E ec o cell cycle synch onisa ion on AdV p oduc ion
125
PART 2
293 cell cycle synch onisa ion in adeno i us p oduc ion
Fe ei a T.B., Pe digão R., Ca ina A.C., Zhang C., Aunins J.G., Ca ondo
M.J.T., Al es P.M. Bio echnol. Bioeng. (submi ed)
Chap e IV.2
126
Abs ac
As he ma ke equi emen s o adeno i us ec o s (AdV) inc ease, he
maximisa ion o he i us p oduc i i y pe cul u e olume pe uni ime is a
key equi emen . Howe e , despi e he ac ha 293 cells can g ow up o
8×106 cell/ml in simple ba ch mode ope a ions, o op imal AdV in ec ion a
maximum cell densi y o 1×106 cell/ml a in ec ion ime has been epo ed.
In addi ion, AdV p oduc ion appea s o be dependen o he cell cycle
phase a he ime o in ec ion. To e alua e he dependence o AdV
p oduc ion on cell cycle phase, 293 cells we e chemically synch onised a
each phase o he cell cycle; a 2.6 old inc ease on cell speci ic
p oduc i i y was ob ained when he pe cen age o cells a he S phase o
he cell cycle was inc eased om 36% o 47%. A ma hema ical equa ion
was used o ela e AdV p oduc i i ies wi h S phase cell synch onisa ion
using his da a.
A empe a u e shi s a egy was also used a emp ed o synch onisa ion
a he S phase, a oiding he use o chemical inhibi o s. S phase
synch onisa ion was possible by dec easing he cul u e empe a u e o 31ºC
du ing 67h and es o ing i o 37ºC du ing 72h. By using his s a egy we
we e able o synch onise 57% o he popula ion in he S phase o he cell
cycle ob aining an inc ease o 7.3 old on cell speci ic p oduc i i y a e
in ec ion.
E ec o cell cycle synch onisa ion on AdV p oduc ion
127
CONTENTS
1. INTRODUCTION________________________________________ 128
2. MATERIALS AND METHODS _______________________________ 130
2.1. Cell line and medium _______________________________ 130
2.2. Adeno i al Vec o __________________________________ 131
2.3. Chemical synch onisa ion o 293 cells __________________ 131
2.4. In ec ion o chemically synch onised 293 cells____________ 132
2.5. Tempe a u e Synch onisa ion o 293 cells _______________ 133
2.6. In ec ion o 293 cells synch onised by empe a u e shi ____ 133
2.7. Flow cy ome ic analysis_____________________________ 134
2.8. Vi us cul u e samples p epa a ion _____________________ 134
2.9. Adeno i al ec o i a ion ___________________________ 134
2.10. Cell concen a ion and iabili y de e mina ion __________ 135
3. RESULTS AND DISCUSSION _______________________________ 135
3.1. 293 cell synch onisa ion wi h chemical compounds________ 135
3.2. Adeno i us ec o p oduc ion in chemically synch onised 293
cells ________________________________________________ 137
3.3. Model o adeno i us p oduc ion in chemically synch onised cells
____________________________________________________ 139
3.4. 293 cell synch onisa ion by empe a u e shi ____________ 141
3.5. Adeno i us ec o p oduc ion in synch onised 293 cells by
empe a u e shi ______________________________________ 144
4. CONCLUSIONS_________________________________________ 145
5. ACKNOWLEDGEMENT ___________________________________ 146
6. REFERENCES__________________________________________ 147
Chap e IV.2
128
1. INTRODUCTION
Adeno i uses a e leading ec o s o gene ans e , wi h p oduc s in
de elopmen o applica ions anging om cance he apeu ics o
p ophylac ic accines and eplacemen he apies o gene ic de iciencies
( o e iews see Gallo e al. (2005), Fe ei a e al. (2005a) and Mo sy e
al., (1998)); wo ldwide, o e 300 clinical ials using Adeno i us Vec o s
(AdV) ha e al eady been app o ed, ongoing o comple ed, making AdV he
mos used deli e y ec o in clinical ials (www.wiley.co.uk/gene he apy/
clinical).
One o he goals o a comme cial AdV cul i a ion p ocess is o maximize
he i us p oduc i i y pe cul u e olume pe uni ime. The cell densi y a
in ec ion is a e y impo an pa ame e as i impac s on he AdV
olume ic p oduc i i y. Howe e , despi e he ac ha 293 cells can g ow
up o 8×106 cell/ml in simple ba ch mode ope a ions, a maximum in ec ion
cell densi y o 1×106 cell/ml has been epo ed as op imal (Nadeau and
Kamen, 2003; Xie e al., 2003; Kamen and Hen y, 2004; Fe ei a e al.,
2005c; Ma anga e al., 2005). An app oach o main ain cell speci ic
p oduc i i y a inc eased cell densi y consis s o in ec ing he cells a e
medium exchange (Ga nie e al., 1994; Iye e al., 1999; Fe ei a e al.,
2005b). Howe e , his p ocedu e adds he ex a complexi y o a cell
sepa a ion s ep and inc eases in p oduc p oduc ion cos due o medium
exchange. O he app oach consis s in he use o a ed-ba ch s a egy, bu
i s success has been limi ed (Nadeau e al., 1996; Fe ei a e al., 2005b).
By using a non-ammoniagenic medium an inc ease o 5 old on cell speci ic
p oduc i i y could be ob ained a a CCI o 3×106 cell/ml s ill bellow he
expec ed (Fe ei a e al., 2005b).
AdV p oduc ion is usually pe o med assuming ha cul u ed cells ep esen
a uni o m a ge o he i us. Howe e , in ac , cells in cul u e a e no
homogenous, including a collec ion o cells wi h dis inc physiologies. A
majo de e minan o cell physiology in cul u e is he cell cycle wi h i s
E ec o cell cycle synch onisa ion on AdV p oduc ion
129
ou s ages (S ein e al., 1999) including ele a ed p o ein syn hesis (G1 and
G2 phases), ele a ed DNA syn hesis (S phase) and changes in memb ane
a icking, cy oskele al o ganiza ion and cell-cell in e ac ions (M phase).
In Chap e IV, Pa 1 was shown ha AdV p oduc ion seems o be
dependen o he cell cycle phase a he ime o in ec ion: du ing cell
g ow h he highes pe cen age o cells a he S phase o he cell cycle is
p esen a app oxima ely 1×106 cell/ml, which has been shown o be he
op imal concen a ion o AdV in ec ion, dec easing wi h inc easing cell
densi y (Zhang e al., 2006). Fu he mo e, Good um and O nelles (1997)
obse ed ha in synch onously g owing HeLa cells, app oxima ely 75% o
he cells in ec ed du ing S phase, p oduced E1B AdV mu an i us, whe eas
only 10% o he cells in ec ed du ing G1 phase we e able o p oduce i us.
The e o e, i is pe inen o synch onize cells a each phase o he cell
cycle in o de o iden i y he bes phase o in ec ion o inc ease cell
speci ic p oduc i i y a high cell densi ies. Se e al me hods o ob aining
synch onised popula ions o mammalian cells in i o ha e been epo ed
by using gene ic, chemical, en i onmen al and physical s a egies (Da is e
al., 2001). Fo e e sible chemical synch onisa ion se e al compounds a e
a ailable, as hymidine (Law e al., 2006) o G1 phase synch onisa ion,
mimosine (Hughes and Cook, 1996) o la e G1 phase synch onisa ion,
aphidicolin (Jackson, 1995), apamicine (Law e al., 2006) and hyd oxyu ea
(Rosenk anz and Becke , 1973) o synch onisa ion a he G1/S ansi ion
phase, s au ospo ine (Koche and Cleme son, 1991) o G2 phase
synch onisa ion and nocodazole (Ho e al., 2001) o synch onisa ion a he
G2/M ansi ion phase. Howe e , in addi ion o he di icul y in ensu ing
hei elimina ion in he inal p oduc , he use o hese chemicals as
synch onising agen s also a ec s cell iabili y, o en p esen ing se e e
limi a ions o la ge scale p oduc ion pu poses.
Besides chemical synch onisa ion, se e al s udies sugges hypo he mia as a
po en ially use ul ool o manipula e mammalian cell cycle dis ibu ion
(Rao and Engelbe g, 1965; Sisken e al., 1965; Wa anabe and Okada, 1967).

Chap e IV.2
130
Typically, mammalian cells a e g own a 37ºC, mimicking mammalian body
empe a u e. A “sub-op imal” empe a u es (25-33ºC) he e is a
p olonga ion o he cell cycle du a ion as a esul o he empo al dila ion
o he G1, S and M (mi osis) phases, wi h G2 being he leas and M he mos
sensi i e phases o empe a u e shi (Riede and Cole, 2002), unde going
he majo inc ease in du a ion (Rao and Engelbe g, 1965; Sisken e al.,
1965). Howe e , heses occu ences a e e y dependen on he cell line
conside ed. Fo example, incuba ion o human diploid ib oblas s (Enninga
e al., 1984) o o a mouse leukemic cell line (Wa anabe and Okada, 1967)
a 31ºC induced cell cycle a es in G1/G0 phase; on he o he hand, when
HeLa cells we e incuba ed a sub-no mal empe a u es he e was an
accumula ion o cells in he M phase (mi osis) (Rao and Engelbe g, 1965).
In his wo k, we epo on he e ec o he cell cycle on AdV p oduc ion.
Fi s , in o de o es ablish he bes cell cycle phase o AdV in ec ion, 293
cells we e chemically synch onised. Then, due o he inhe en p oblems
associa ed wi h he use o hese chemicals compounds o p oduc ion
pu poses, a synch onisa ion s a egy based on empe a u e shi was
de eloped and e alua ed in e ms o AdV p oduc ion a high cell densi ies
in 2 L bio eac o s.
2. MATERIALS AND METHODS
2.1. Cell line and medium
Ancho age-dependen 293 cells, pu chased om S a agene (Ca alog
#240085), we e cul u ed in Dulbecco’s Modi ied Eagle Medium (DMEM)
supplemen ed wi h 10% ( / ) hea -inac i a ed (56 ºC, 30 min.) Foe al
Bo ine Se um (FBS) (all om In i ogen, Glasgow, UK) and 4.5 g/L o
glucose (Me ck, Da ms ad , Ge many) a a humidi ied a mosphe e o 5%
CO2 in ai a 37ºC. 293 cells we e ou inely p opaga ed wice a week a e
ypsin/EDTA (In i ogen, Glasgow, UK) addi ion a oom empe a u e.
E ec o cell cycle synch onisa ion on AdV p oduc ion
131
Suspension adap ed 293 cells we e g own in CD 293 (In i ogen, Glasgow,
UK), supplemen ed wi h 4 mM o glu ama e (non-ammoniagenic medium)
(Sigma-Ald ich, S . Louis, MO) a a humidi ied a mosphe e o 8% CO2 in ai
a 37ºC in shake lasks (Co ning, NY). Cells we e ou inely p opaga ed wice
a week using an inoculum o 0.5×106 cell/ml.
2.2. Adeno i al Vec o
Recombinan AdV exp essing he GFP p o ein we e gene a ed by
homologous ecombina ion o plasmid and diges ed DNA in E. coli s ain
BJ5183 ( ec+). The GFP shu le ec o was i s cloned in o a ans e
ec o ; he esul ing plasmid was hen linea ized wi h a es ic ion enzyme
(Swa I) and co- ans o med in o E. coli oge he wi h he plasmid
con aining AdV (pKP1.3). Recombinan s we e selec ed wi h ampicillin and
sc eened by es ic ion enzyme analysis. The ecombinan AdV cons uc
was subsequen ly clea ed wi h Pac I o expose i s ITR (In e ed Te minal
Repea s) and ans ec ed in o 293 cells o p oduce i al pa icles.
2.3. Chemical synch onisa ion o 293 cells
Fo chemical synch onisa ion, 293 cells we e seeded in 6 well pla es a
1106 cell/well, wi h a cul u e olume o 2 mL o DMEM supplemen ed wi h
10% ( / ) FBS and 1% ( / ) an ibio ic solu ion (10000 µg/mL s ep omycin
and 10000 U/mL penicillin) (Sigma-Ald ich, S . Louis, MO). One day a e ,
seeded cells we e incuba ed wi h each inhibi o a h ee di e en
concen a ions du ing 6, 12, 24 and 48h. A con ol o each inhibi o whe e
only he espec i e sol en was added o he cell cul u e was pe o med.
The expe imen al design is summa ised in Table 4.2.1. A e he incuba ion
pe iod, 293 cells we e diges ed a oom empe a u e wi h ypsin/EDTA
and analysed by low cy ome y.
Chap e IV.2
132
Table 4.2.1. Re e sible chemical inhibi o s used and co esponden cell cycle
a es poin . S ock solu ions, es ed concen a ions and con ol expe imen s a e
shown o each case.
Inhibi o
Type
Ta ge
A es Poin
Inhibi o S ock
solu ion
Tes ed
concen a ions Con ol
Mimosine La e G1
100 mM in a PBS
solu ion 0.1%
( / ) in DMSO
1 mM
1 M
1 nM
PBS 0.1%
( / ) in
DMSO
Hyd oxyu ea G1/S
ansi ion
100 mM in a PBS
solu ion 0.1%
( / ) in DMSO
1 mM
1 M
1 nM
PBS 0.1%
( / ) in
DMSO
Aphidicolin G1/S
ansi ion 2.96 mM in DMSO
0.1 mM
1 M
1 nM
DMSO
S au ospo ine G2 0.214 mM in
e hanol 96% ( / )
10 mM
1 M
0.1 M
E hanol
96% ( / )
Nocodazole G2/M
ansi ion
13.3 mM in a PBS
solu ion 0.1%
( / ) in DMSO
1 mM
1 M
1 nM
PBS 0.1%
( / ) in
DMSO
2.4. In ec ion o chemically synch onised 293 cells
In ec ion o chemically synch onised cells was pe o med by seeding 293
cells in 6 well pla es a 1106 cell/well, wi h a cul u e olume o 2 mL o
DMEM supplemen ed wi h 10% ( / ) FBS and 1% ( / ) an ibio ic solu ion
(10000 µg/mL s ep omycin and 10000 U/mL penicillin). One day a e , he
bes inhibi o y condi ions, de e mined be o ehand, we e added: 1 mM
hyd oxyu ea du ing 48h, 0.1 mM aphidicolin du ing 12h and 1 µM
s au ospo ine du ing 48h. In he expe imen al con ols only he sol en s
we e added. A e he incuba ion pe iod, cells we e cen i uged a 200 g
du ing 10 min. a 4ºC and suspended in 500 µL o esh DMEM wi hou FBS
E ec o cell cycle synch onisa ion on AdV p oduc ion
133
and eseeded in a new well. Then, cells we e in ec ed wi h AdV using a
mul iplici y o in ec ion (MOI) o 10. 30 min. a e wa ds, 1 mL o DMEM
supplemen ed wi h 10% ( / ) FBS and 1% ( / ) an ibio ic solu ion (10000
µg/mL s ep omycin and 10000 U/mL penicillin) was added o each well.
In ec ed 293 cells we e hen ha es ed a se e al imes a e diges ion a
oom empe a u e wi h ypsin/EDTA.
2.5. Tempe a u e Synch onisa ion o 293 cells
Fo empe a u e synch onisa ion, 293 cells adap ed o g ow in suspension
we e inocula ed a 0.5×106 cell/ml in 125 mL shake lasks wi h a wo king
olume o 40 mL in a humidi ied a mosphe e o 8% CO2 in ai a 37ºC. Once
he concen a ion o 2×106 cell/ml cells was eached, incuba ion a 29, 31,
33, 35 e 37ºC was ca ied ou o 72h, and he cell cycle ollowed by low
cy ome y.
2.6. In ec ion o 293 cells synch onised by empe a u e shi
In ec ion o 293 cells synch onised by empe a u e shi was done in 2 L
bio eac o s (B aun, Melsungen, Ge many). Fo his pu pose, wo
bio eac o s we e inocula ed a 0.5×106 cell/ml wi h 293 cells adap ed o
g ow in suspension. Once he popula ion eached he concen a ion o
3×106 cell/ml, one o he bio eac o s (con ol) was in ec ed and he o he
was synch onised a he S phase o he cell cycle and in ec ed. All he
in ec ions we e done using a MOI o 10 and AdV ha es ed a 48 hpi.
Synch onisa ion was pe o med by i s ly lowe ing he cul u e empe a u e
o 31ºC du ing 67h and hen es o ing i o 37ºC du ing 72h. The agi a ion
a e was main ained a 110 pm (NRe = 6800); pH was con olled a 7.2 by
ae a ion wi h a CO2 gas-mix u e and NaOH 0.2 M; he dissol ed oxygen was
con olled a 80% ai sa u a ion.