The effect of the cell death suppressor vMIA on the production of a recombinant protein in the adenovirus-293 expression system

Protein Expression and Purification 64 (2009) 179–184

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Protein Expression and Purification

journal homepage: www.elsevier .com/locate /yprep

The effect of the cell death suppressor vMIA on the productionof a recombinant protein in the adenovirus-293 expression system

Helena L.A. Vieira a, Liliana Cunha a, Victor S. Goldmacher b, Paula M. Alves a,*

a Instituto de Tecnologia Química e Biológica (ITQB-UNL), Instituto de Biologia Experimental e Tecnológica (IBET), Apartado 12, 2781-901 Oeiras, Portugalb ImmunoGen, Inc., 830 Winter Street, Waltham, MA 02451, USA

a r t i c l e i n f o

Article history:Received 18 September 2008and in revised form 27 October 2008Available online 18 November 2008

Keywords:Recombinant protein productionAdenovirusApoptosisvMIACell death suppressorTechnology

1046-5928/$ - see front matter � 2008 Elsevier Inc. Adoi:10.1016/j.pep.2008.11.004

* Corresponding author. Fax: +351 214 421 161.E-mail address: [email protected] (P.M. Alves).

a b s t r a c t

Apoptosis is a major problem in animal cell cultures during production of biopharmaceuticals, such asrecombinant proteins or viral vectors. A 293 cell line constitutively expressing vMIA (viral mitochon-dria-localized inhibitor of apoptosis) was constructed and examined on production of a model recombi-nant protein, green fluorescent protein (GFP) in the adenovirus-293 expression system, and onproduction of a model infectious adenoviral vector. vMIA-293 cells were more resistant than the parental293 cells to apoptosis induced by either oxidative stress, or by adenovirus infection. The yield of GFP pro-duced in vMIA-293 cell cultures was consistently higher (�140%) compared to that in the parental cells.vMIA reduced production of adenovirus infectious particles, which was not due to a decline of adenovirusreplication, since adenoviral DNA replication rate in vMIA-293 cells was higher than that in the parentalcells.

In conclusion, introduction of the vMIA gene into the 293 cell line is a promising strategy to improverecombinant protein production in the adenovirus-293 expression system.

� 2008 Elsevier Inc. All rights reserved.

1

Introduction

Optimization of bioprocesses is essential for cost-effective pro-duction of biopharmaceuticals, such as recombinant proteins,viruses and cells. Cell death in bioreactors represents a major prob-lem in animal cell culture technology because it decreases the glo-bal productivity yield [1,2]. Several factors have been implicated indecreasing the cell viability in bioreactors: nutrient depletion,shear stress, hypoxia, accumulation of toxic metabolites andbyproducts, and apoptosis induced by viral infections [3].

Two main approaches to improve cell viability in bioreactorsare: (i) optimization of the extracellular environment to improvecell nutrition, the levels of growth hormones, optimize the pHand reduce the levels of toxic metabolites, and thus reduce physi-ological stress, and (ii) direct targeting of apoptotic pathwaysthrough either genetic modification of the cell line, or addition ofanti-apoptotic chemicals, proteins or peptides to the medium(see [3] for a review). For example, fortification of medium withinsulin and transferrin suppressed death of CHO cells in serum freemedia [4]. Inhibitors of caspases enhanced the viability of CHO and293 cell cultures [5]. Addition of the anti-oxidant N-acetylcysteineincreased the lifespan of the rat prostate carcinoma cell line AT3,following its infection with a Sindbis virus vector [6]. Addition of

ll rights reserved.

catalase which destroys hydrogen peroxide into a baculovirus-in-sect cell system suppressed cell death and improved recombinantprotein production [7]. The media supplementation with hemo-lymph enhanced insect cell culture longevity [8].

At the level of genetic manipulation, various strategies were ap-plied to limit cell death. Overexpression of the anti-apoptotic Bcl-2family members, such as Bcl-2 and Bcl-XL, was one of the most fre-quently used approach to suppress apoptosis (see [9] for review). Itwas tested on several cell lines: myeloma NS0, hybridoma cells orCHO [10–13]. The overexpression of human telomerase reversetranscriptase (hTERT)1 was reported to decrease apoptosis inCHO K1 cells [14]. Another approach, still not extensively exam-ined, is the expression of anti-apoptotic viral proteins. The adeno-viral E1B-19K cell death suppressor reduced apoptosis in BHK andCHO cells [15,16]. The Epstein–Barr virus Bcl-2 homologue bhrf-1protected hybridoma cell lines against cell death [17].

Adenoviruses are attractive vectors for gene therapy and vacci-nation because of their high efficiency of gene transfer in a broadspectrum of cell types, as well as for high levels of recombinantprotein production [18,19], including production of proteins thatare toxic for the producer cell lines [20,21].

Abbreviations used: hTERT, human telomerase reverse transcriptase; vMIA, viralmitochondria-localized inhibitor of apoptosis; ANT, adenine nucleotide translocator;MEM, minimum essential medium; FBS, foetal bovine serum; GFP, green fluorescentprotein; MOI, multiplicity of infection; PCR, Polymerase chain reaction; ECL, enhancedchemiluminescence;

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In this study, we examined the ability of the powerful anti-apoptotic viral protein vMIA (viral mitochondria-localized inhibi-tor of apoptosis) to limit cell death and to improve production ofa model protein and of a model adenoviral vector in the Adenovi-rus-293 expressing system. vMIA is encoded by UL37 exon 1 of hu-man cytomegalovirus and is predominantly localized inmitochondria, where it forms a complex with adenine nucleotidetranslocator (ANT) [22,23] and suppresses cell death by sequester-ing Bax in an inactive form at mitochondria [24,25].

Although the mechanism of the cell death suppression by vMIAis fairly well understood [26,27], this viral protein has not yet beenexploited to improve bioprocesses.

Materials and methods

Cells and media

Anchorage-dependent 293 cells, purchased from ATCC (ATCC-CRL-1573), were routinely cultured in Minimum Essential Medium(MEM) supplemented with 5% (v/v) heat-inactivated (56 �C,30 min) foetal bovine serum (FBS), 2 mM glutamine in an humidi-fied atmosphere of 5% CO2 in air at 37 �C.

Plasmids, transfections and drug selection

Mammalian expression vector pcDNA3myc, and vMIAmyc/pcDNA3 which encodes vMIA fused with three tandem copies ofthe human c-myc epitope at its C terminus was described previ-ously (Goldmacher, 1999).

For stable transfection, 293 cells were seeded in 6-well plates at6 � 105 cells/well, 2 days later cells were transfected with 2 lg ofvMIAmyc/pcDNA3 by the calcium phosphate precipitation method[28]. Two days later, cells were three fold diluted and cultured un-der a selection pressure of 500 lg/mL Geneticin (Invitrogen, Glas-gow, UK) for 15 days. Cell cloning was performed by the limitingdilution method. Cells were seeded at 1 cell per well in 96-wellplates with 50% conditioned medium (medium that was previouslyused in 293 cell culture for 2 days and filtered through 0.45 lm toeliminate suspended cells) containing 20% FBS and 500 lg/mLGeneticin. Twenty clones were picked and analysed.

Adenovirus and infection

A replication-defective adenovirus (AdV) derived from type 5AdV, coding for green fluorescent protein (GFP) was produced as de-scribed in (Ferreira et al., 2008). 293 or vMIA-293 cells were seededin 24-well plates at 0.25 � 106 cells/well. Twelve hours later cellswere infected with AdV at a multiplicity of infection (MOI) of 10,in 0.5 mL of non-supplemented MEM medium and incubated for30 min at 37 �C. Then 0.5 mL of supplemented medium was addedto each well. Samples from individual wells were harvested at 24,48, 72, 96 and 120 hours post-infection (hpi) for apoptosis-associ-ated parameters assessment, virus titration, real time quantitativePCR (for virus replication analysis) and GFP recombinant proteinquantification.

Polymerase chain reaction

Genomic DNA was extracted from 293 and vMIA-293 cells using‘‘High Pure PCR Template Preparation Kit” (Roche Diagnostics,Mannheim, Germany). Polymerase chain reaction (PCR) was per-formed using specific forward and reverse primes designed forvMIA gene: 50-GTC TCC AGT CTA CGT GAA TC-30 and 50-GTT GTGCTG CAG CAT CCG AG-30, respectively. Denaturation program was95 �C for 40, followed by 35 cycles of 95 �C for 10300 0, 55 �C for 10

and 72 �C for 10. DNA was analysed by electrophoresis in 1% aga-rose gels and using standard procedures.

Western blot

Western blot techniques were used to verify the presence ofvMIA protein in the constructed cell lines and to quantify GFPrecombinant protein production after adenovirus infection. The to-tal protein in the samples was quantified by MicroBCATM ProteinAssay Kit (Pierce, Rockford, IL, USA) to ensure equal loading ofgel lanes, reduced and resolved by denaturating electrophoresison a 1 mm NuPAGE� Novex BIS-Tris Gel (Invitrogen, UK) and elec-trically transferred to a nitrocellulose membrane (HybondTM-C ex-tra, Amersham Biosciences). vMIAmyc protein was stained with amonoclonal anti-myc (Santa Cruz Biotechnology, INC), and GFPprotein was stained with a monoclonal anti-GFP (Sigma). Blotswere developed using the ECL (enhanced chemiluminescence)detection system after incubation with HRP-labeled anti-mouseIgG antibody (Amersham Biosciences, UK). The area and intensityof bands were quantified by densitometry analysis (GraphPadPrism 4), and are presented as arbitrary units per number of in-fected cell (AU/cell).

Apoptosis induction

Apoptosis by oxidative stress was induced by exposure of cellsto tert-butylhydroperoxide, t-BHP (Sigma) for 4 h in the range ofconcentrations of 40–120 lM. Apoptotic cell death was also in-duced by adenovirus infection as described above.

Flow cytometry

A flow cytometer (Partec, Germany) was used to analyse apop-tosis-associated parameters and the levels of intracellular GFP re-combinant protein. This cytometer contains a blue solid statelaser (488 nm) with FL1 green fluorescence channel for DiOC andGFP detection at 530 nm, and a FL3 red fluorescence channel forPI detection at 650 nm. To detect apoptosis induced by oxidativestress, or by adenoviral vector infection, cell samples were col-lected by trypsinisation after a 4-h treatment with t-BHP, or aftera 24–96 hpi, respectively. Intact cells were gated by the forwardand side scatter. Two fluorochromes were used, 3,3’-dihexyloxa-carbocyanine iodide (DiOC6(3), 20 nM) to quantify the mitochon-drial transmembrane potential (DWm) (ref.), and propidiumiodide (PI, 1 lg/ml) to determine cell viability, based on the plasmamembrane integrity (ref.). In addition, the percentage of cellsexpressing GFP after infection with the adenoviral vector wasdetermined. The acquisition and analysis of the results was per-formed with FlowMax� (Partec) software.

Fluorescent microscopy

0.25 � 106 cells of 293 or vMIA-293 cell line were plated into13 mm-diameter coverslips and cultured for 24 h, and then ex-posed for 4 h to tert-butylhydroperoxide (Sigma). The cells werethen stained with Hoechst 33342 (2 lV, Sigma), and followed byfluorescence microscopic assessment of apoptotic nuclei. Cellswere observed on a Leica DMRB microscope using a filter cube pre-senting UV excitation range with a bandpass of 340–380 nm ofwave length.

Titration of AdV

Anchorage-dependent 293 cells were seeded in 24 well plates at0.25 � 106 cell/ml. Twenty-four hours later, cells were infectedwith 1 ml of serial dilutions of AdV in infection medium

Fig. 1. Detection of vMIA recombinant protein in the new vMIA-293 cell line. vMIArecombinant protein was detected in extracts of vMIA-293 cells by Western blotusing a monoclonal anti-myc, which detects the target sequence myc fused withvMIA. Lane 1 corresponds to control 293 cell line, while lane 2 corresponds to vMIA-293 cell line.

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(MEM + 5% FBS). After 24 h of incubation at 37 �C cells were har-vested by trypsinization and immediately analysed by flow cytom-etry. The green fluorescence signal was collected by aphotomultiplier tube after passing through a 525 (±20) nm bandpass filter (FL1). GFP fluorescence of 3 � 104 single viable cellsper sample selected on forward-angle light scattering (FS) versusside-angle light scattering (SS) scatter basis were analysed andhighly fluorescent cells were gated. The cytometric parameterswere set to provide accurate discrimination between non-fluores-cent negative cells and positive GFP-fluorescence cells on a FL1versus FSC density plot in order to estimate the proportion of in-fected cells: One fluorescent cell corresponds to one infectious par-ticle (ip) when less than 30% of the cells are fluorescent [29]. Dataanalysis was performed using FlowMax� software.

Quantitative real time PCR

For the quantitative real time PCR a conserved sequence of ade-novirus type 5 was used to quantify adenovirus [30]. DNA amplifi-cation was done using the LightCycler� system with ‘‘Fast StartMaster SYBR Green I kit” (Roche Diagnostics, Germany). For a20 ll PCR reaction, 2 ll DNA template and 18 ll of Mastermixwere added to each capillary. The Mastermix was prepared to ob-tain a final concentration of 3 mM MgCl2 and 0.5 lM of each pri-mer in each capillary. The oligonucleotide primers used were:forward primer (5-TCG TAG AAG TGG ACG GT-3) and reverse pri-mer (5-GTG TTC GTG ACT GAA GC-3). The primers were designedto blast only the gene of interest. AdV DNA was denatured for10 min at 95 �C. The standard amplification program included 30three-step cycles: (i) denaturation-heating at 95 �C for 1 s, (ii)annealing–cooling at 56 �C during 5 s, and (iii) elongation-heatingat 72 �C for 10 s. Fluorescence was acquired at the end of the elon-gation step of each cycle. Data were analysed using the Light-Cycler� software.

Data analysis

The kinetics of adenovirus replication can be directly correlatedwith viral DNA replication rate (assessed by Q-PCR described pre-viously), which follows a first order kinetics pattern [31]. The evo-lution profile characteristic of adenovirus DNA replication cantherefore be described by equation:

V ¼ V0 � expðlr � tÞ

where V is viral DNA copies per ml, V0 is the initial viral DNA copiesper ml, t is time (hpi) and lr is the DNA replication rate constant(hpi�1), which is calculated to compare distinct kinetics of adenovi-rus replication.

Fig. 2. Clone selection by anti-apoptotic activity of vMIA. Apoptosis in parental 293 cellsprotein was triggered by oxidative stress induced by a 4-h exposure of cells to the oxidanmembrane and high mitochondrial potential, as detected by flow cytometry, using PI an

Results and discussion

vMIA-293 cell line construction

Human 293 cells were stably transfected with vMIAmyc/pcDNA3. After antibiotic selection, 20 clones were picked and ana-lysed for the presence of vMIA DNA sequence, which was checkedby PCR (data not shown), as well as for the expression of vMIAmycrecombinant protein, which was detected by Western blot. Fig. 1shows an example of recombinant vMIAmyc expression in a typicalclone. 15 clones were positive for the presence of vMIA DNA se-quence and for the expression of this recombinant protein.

vMIA confers protection against apoptosis induced by oxidative stress

Viral infections induce oxidative stress and apoptosis of the hostcell [32,33]. We tested if 293 cell clones expressing vMIA aquiredresistance to the oxidative stress-related apoptosis induced bythe oxidant tert-butylhydroperoxide.

Cell viability, as detected by exclusion of propidium iodide, andthe degree of apoptosis as detected by the dissipated mitochon-drial transmembrane potential (DWm), using DiOC6(3) as a fluoro-chrome, were assessed by flow cytometry. We then determined thepercentage of viable cells as those with both the high mitochon-drial potential and not stained with propidium iodide. The 15 pre-viously positive clones were treated with tert-butylhydroperoxideat 80 lM during 4 h and cell viability was assessed; 8 clones weremore resistant to apoptosis then control, while 7 clones presentedsimilar response to oxidative stress as control 293 cells (Fig. 2).Fig. 3A shows a representative result of t-BHP dose response fora typical clone (designed as vMIA-293 cell line, clone 1 fromFig. 2) after 4 h of treatment.

Another consequence of apoptotic cell death is the nuclearchromatin condensation, which can be detected by fluorescentmicroscopy, using Hoechst 33324 [23]. We found that the 293clones that constitutively expressed vMIA, aquired resistance tochromatin condensation following their exposure to t-BHP (theexperiment with vMIA-293 cells is shown in Fig. 3B). Taken to-

and in 15 different clones containing vMIA DNA and expressing vMIA recombinantt t-BHP, at 80 lM. The percentage of viability corresponds to cells with intact plasmad DiOC as fluorochromes, respectively.

Fig. 4. vMIA provides protection against apoptosis induced by adenovirus infection.293 and vMIA-293 cells were infected with adenovirus at MOI of 10, cell viabilitywas followed for the period of 4 days post infection. Cell viability was determinedby flow cytometry using PI for detection of plasma membrane integrity.

Fig. 5. vMIA increases GFP producing cells. 293 and vMIA-293 cells were infectedwith adenovirus coding for green fluorescent protein at MOI of 10, and thepopulations of GFP expressing cells were examined by flow cytometry. In (A) thepercentage of GFP expressing cells is relative to the total amount of cells in theculture, for 293 and vMIA-293 cells. (B) Displays the percentage of GFP expressingcells among the viable cells in the 293 and vMIA-293 populations. Cell viability wasdetermined by flow cytometry using PI for detection of plasma membrane integrity.

Fig. 3. vMIA confers protection against apoptosis induced by oxidative stress.Apoptosis in parental 293 and vMIA-293 cells was triggered by oxidative stressinduced by a 4-h exposure to the oxidant t-BHP (from 0 to 120 lM). In (A) thepercentage of viability corresponds to cells with intact plasma membrane and highmitochondrial potential, as detected by flow cytometry, using PI and DiOC asfluorochromes, respectively. In (B), apoptosis was monitored by fluorescentmicroscopy. Normal nuclei (of viable cells) versus apoptotic nuclei (chromatincondensation analysis) were assessed using the DNA binding dye Hoechst.

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gether, these data indicate that vMIA prevents cell death inducedby oxidative stress in 293 cells. We then chose a typical clone, de-signed vMIA-293, as a candidate to improve adenovirus-293expression system for production of viral vectors and/or recombi-nant proteins.

Apoptosis induction by adenovirus infection is delayed by vMIA

Since infection of 293 cells with the adenoviral vector AdV in-duces host cell death, we tested the cell death suppressing activityof vMIA in cells infected with AdV. vMIA-293 cells and the parental293 cells were infected with AdV at a MOI of 10, and their viability,during a 4-day culturing after the infection, was examined by thepropidium iodide exclusion test on a flow cytometer. As shownin Fig. 4, vMIA-293 cells were viable for a longer period of time(approximately a 1-day-delay) than the parental cells. These datasuggest that vMIA prolongs 293 cell life span after infection withthe adenoviral vector.

Production of GFP recombinant protein in vMIA-293 cells

Having established that vMIA-293 cells are more resistant toboth oxidative stress induced and AdV-infection-induced apopto-sis, we set out to examine if this cell line would be a better cell lineto produce a model recombinant protein, GFP, or to produce amodel adenoviral vector, AdV. Cells were infected with AdV, anadenoviral expression vector that encodes GFP, and the percentageof GFP-expressing cells in the total cell population was examinedby flow-cytometry. We found that over the period of 4 days afterinfection, the percentage of GFP expression in vMIA-293 cells

was significantly higher than that in the parental 293 cells(Fig. 5A). For example, at 72 hpi 72% of vMIA-293 cells expressedGFP as compared to 42%, of 293 cells. In order to determine if theincrease in GFP expressing cells was only due to the higher viabil-ity of vMIA-expressing cells, or if there was any additional effect ofvMIA on the recombinant protein production, we compared thelevels of GFP expressing cells within viable vMIA-293 cells and via-ble parental 293 cells (Fig. 5B). Again at 72 hpi 87% of vMIA-293cells expressed GFP as compared to 45% of 293 cells. Therefore,vMIA improves GFP production by prolonging cell viability andalso, apparently, by increasing the producer cell population.

Fig. 6. vMIA enhances specific productivity of recombinant protein. 293 and vMIA-293 cells were infected by AdV at MOI of 10. Culture extracts were harvested at 24,48 and 72 hpi to quantify recombinant protein production. Specific GFP productionwas indirectly assessed by Western blot densitometry using a monoclonal anti-GFP.

Fig. 8. Effect of vMIA on adenovirus replication. Viral DNA replication rate(assessed by Q-PCR) can be correlated with the kinetics of adenovirus replication.In order to address adenoviral replication in the presence or absence of vMIA,several samples (one each 5 h, up to 30 h post-infection) were harvested, viral DNAwas purified and quantitative real time PCR was performed. Adenovirus DNAreplication can be described by equation: V = V0 � exp(lr � t), where V is viral DNAcopies per ml, V0 is the initial viral DNA copies per ml, t is time (hpi) and lr is theDNA replication rate constant (hpi�1), which is calculated to compare distinctkinetics of adenovirus replication.

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The specific production of GFP recombinant protein in vMIA-293 cells and in the parental 293 cells at 24, 48 and 72 hpi wascompared by Western blot densitometry (Fig. 6). The productionof GFP in vMIA-293 cells was consistently higher (�140%) over thisperiod of time compared to that in 293 cells.

Production of the adenoviral vector AdV in vMIA-293 cells

We also compared vMIA-293 cells and the parental 293 cells onthe production of AdV infectious particles. Both cell lines were in-fected with several concentrations of AdV, 24 h later cells wereanalysed by flow cytometry and the amount of infected cells(GFP expressing cells) was assessed to quantify the AdV infectiousparticles. We found (Fig. 7) that vMIA-293 cells produce compara-ble (slightly lower) amounts of adenovirus infectious particles than293 cells.

In order to understand better why adenovirus production invMIA-293 cells was not higher than that in 293 cells, we examinedthe time-course of AdV viral DNA replication in these cell linesupon their infection with AdV. This was assessed by real-timequantitative polymerase chain reaction (Q-PCR) technique. The re-sults are shown in Fig. 8. We found that the DNA replication rateconstants for 293 cells and for vMIA-293 cells, l(293) were

Fig. 7. Effect of vMIA on the production of adenoviral infectious particles. 293 andvMIA-293 cells were infected by adenoviral vectors at MOI of 10, samples weretaken at 24, 48, 72, 96 and 120 hpi to be titrated later. For adenovirus titration,several dilutions of each sample were used to infect 293 cells, 24 hours laterinfected cells were assessed by flow cytometry based on fluorescent emission ofGFP protein coded by the adenoviral vector. According to [29] one fluorescent cellcorresponds to one infectious particle (ip) when less than 30% of the cells arefluorescent.

0.13 ± 0.02, and 0.18 ± 0.02, respectively, suggesting that the viralDNA replication rate in vMIA-293 cells was not lower than thatin 293 cells. On the basis of this result, we speculate that the pro-duction of the infectious AdV particles is reduced not at the stageof viral DNA replication, but, perhaps, at a later stage of virus for-mation/maturation. Further studies will be needed to examine therole of vMIA in the interactions between host cell and virus duringviral maturation and formation of infectious viral particles.

Conclusions

In this study we have constructed a new 293 cell line expressinga powerful anti-apoptotic viral protein vMIA. The presence of vMIAincreased 293 cell viability and improved the production of a mod-el recombinant protein (GFP), however, this anti-apoptotic proteinis not able to augment the productivity of a model adenoviral vec-tor, AdV. In conclusion, the new vMIA-293 cell line represents a no-vel and promising strategy to improve recombinant proteinproduction based on adenovirus-293 expression system.

Acknowledgments

This work was supported by Fundação para a Ciência e Tecnolo-gia, Portugal, for HLAV’s SFRH/BPD/14575/2003 fellowship. Theauthors express their gratitude to Tiago B. Ferreira and CláudiaS.F. Queiroga for technical support.

References

[1] M. Al-Rubeai, R.P. Singh, Apoptosis in cell culture, Curr. Opin. Biotechnol. 2(1998) 152–156.

[2] M. Butler, Animal cell cultures: recent achievements and perspectives in theproduction of biopharmaceuticals, Appl. Microbiol. Biotechnol. 68 (2005) 283–291.

[3] N. Arden, M.J. Betenbaugh, Life and death in mammalian cell culture: strategiesfor apoptosis inhibition, Trends Biotechnol. 22 (2004) 174–180.

[4] N.A. Sunstrom et al., Insulin-like growth factor-I and transferrin mediategrowth and survival of Chinese hamster ovary cells, Biotechnol. Prog. 16 (2000)698–702.

[5] T.M. Sauerwald, G.A. Oyler, M.J. Betenbaugh, Study of caspase inhibitors forlimiting death in mammalian cell culture, Biotechnol. Bioeng. 81 (2003) 329–340.

[6] A.J. Mastrangelo et al., Antiapoptosis chemicals prolong productive lifetimes ofmammalian cells upon Sindbis virus vector infection, Biotechnol. Bioeng. 65(1999) 298–305.

184 H.L.A. Vieira et al. / Protein Expression and Purification 64 (2009) 179–184

[7] H.L. Vieira et al., Catalase effect on cell death for the improvement ofrecombinant protein production in baculovirus-insect cell system, BioprocessBiosyst. Eng. 29 (2006) 409–414.

[8] L. Maranga et al., Enhancement of Sf-9 cell growth and longevity throughsupplementation of culture medium with hemolymph, Biotechnol. Prog. 19(2003) 58–63.

[9] B.S. Majors, M.J. Betenbaugh, G.G. Chiang, Links between metabolism andapoptosis in mammalian cells: applications for anti-apoptosis engineering,Metab. Eng. 9 (2007) 317–326.

[10] K.C. O’Connor et al., Modeling suppression of cell death by Bcl-2 over-expression in myeloma NS0 6A1 cells, Biotechnol. Lett. 28 (2006) 1919–1924.

[11] V. Ifandi, M. Al-Rubeai, Regulation of cell proliferation and apoptosis in CHO-K1 cells by the coexpression of c-Myc and Bcl-2, Biotechnol. Prog. 21 (2005)671–677.

[12] B.T. Tey et al., Influence of bcl-2 on cell death during the cultivation of aChinese hamster ovary cell line expressing a chimeric antibody, Biotechnol.Bioeng. 68 (2000) 31–43.

[13] N.H. Simpson et al., In hybridoma cultures, deprivation of any single aminoacid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene, Biotechnol. Bioeng. 59 (1998) 90–98.

[14] F. Crea et al., Over-expression of hTERT in CHO K1 results in decreasedapoptosis and reduced serum dependency, J. Biotechnol. 121 (2006) 109–123.

[15] T. Nivitchanyong et al., Anti-apoptotic genes Aven and E1B–19K enhanceperformance of BHK cells engineered to express recombinant factor VIII inbatch and low perfusion cell culture, Biotechnol. Bioeng. 98 (2007) 825–841.

[16] B. Figueroa Jr. et al., Enhanced cell culture performance using inducible anti-apoptotic genes E1B–19K and Aven in the production of a monoclonal antibodywith Chinese hamster ovary cells, Biotechnol. Bioeng. 97 (2007) 877–892.

[17] A. Nilou, M.J. Betenbaugh, Life and death in mammalian cell culture: strategiesfor apoptosis inhibition, Trends Biotechnol. 22 (2004) 174–180.

[18] T.B. Ferreira et al., Use of adenoviral vectors as veterinary vaccines, Gene Ther.12 (2005) S73–S83.

[19] F. Wurm, A. Bernard, Large-scale transient expression in mammalian cells forrecombinant protein production, Curr. Opin. Biotechnol. 10 (1999) 156–159.

[20] J. Cote et al., Serum-free production of recombinant proteins and adenoviralvectors by 293SF-3F6 cells, Biotechnol. Bioeng. 59 (1998) 567–575.

[21] A. Garnier et al., Scale-up of the adenovirus expression system for theproduction of recombinant protein in human 293S cells, Cytotechnology 15(1994) 145–155.

[22] V.S. Goldmacher et al., A cytomegalovirus-encoded mitochondria-localizedinhibitor of apoptosis structurally unrelated to Bcl-2, Proc. Natl. Acad. Sci. USA96 (1999) 12536–12541.

[23] H.L. Vieira et al., The adenine nucleotide translocator: a target of nitric oxide,peroxynitrite, and 4-hydroxynonenal, Oncogene 20 (2001) 4305–4316.

[24] D. Arnoult et al., Cytomegalovirus cell death suppressor vMIA blocks Bax- butnot Bak-mediated apoptosis by binding and sequestering Bax at mitochondria,Proc. Natl. Acad. Sci. USA 101 (2004) 7988–7993.

[25] A.L. Pauleau et al., Structure-function analysis of the interaction between Baxand the cytomegalovirus-encoded protein vMIA, Oncogene 26 (2007) 7067–7080.

[26] V.S. Goldmacher, Cell death suppression by cytomegaloviruses, Apoptosis 10(2005) 251–265.

[27] V.S. Goldmacher, vMIA a viral inhibitor of apoptosis targeting mitochondria,Biochimie 84 (2002) 177–185.

[28] J. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual, C.S.H.Laboratory, Editor. 1989: New York.

[29] I. Nadeau et al., 293SF metabolic flux analysis during cell growth and infectionwith an adenovirual vector, Biotechnol. Prog. 16 (2000) 872–884.

[30] L. Ma et al., Rapid determination of adenoviral vector titers by quantitativereal-time PCR, J. Virol. Methods 93 (2001) 181–188.

[31] H.L. Vieira et al., Triple layered rotavirus VLP production: kinetics of vectorreplication, mRNA stability and recombinant protein production, J. Biotechnol.120 (2005) 72–82.

[32] T. Akaike, Role of free radicals in viral pathogenesis and mutation, Rev. Med.Virol. 11 (2001) 87–101.

[33] R. Kannan et al., Impairment of conjunctival glutathione secretion and iontransport by oxidative stress in an adenovirus type 5 ocular infection model ofpigmented rabbits, Free Radic. Biol. Med. 37 (2004) 229–238.