DOI: 10.1002/bit.20871 Abstract: Based on the transcriptional profiling of CHO cell culture using microarray, four key early apoptosis signaling genes, Fadd, Faim, Alg-2, and Requiem, we
Trang 1Targeting Early Apoptotic Genes in Batch
and Fed-Batch CHO Cell Cultures
Danny Chee Furng Wong,1,2Kathy Tin Kam Wong,1Peter Morin Nissom,1
Chew Kiat Heng,2Miranda Gek Sim Yap1,3
1Bioprocessing Technology Institute, Biomedical Sciences Institutes,
Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way,
#06-01, Centros, Singapore 138668; telephone:þ65-6478-8880; fax: þ65-6478-9561;
e-mail: miranda_yap@bti.a-star.edu.sg
2Department of Paediatrics, National University of Singapore, Singapore
3Department of Chemical and Biomolecular Engineering,
National University of Singapore, Singapore
Received 2 June 2005; accepted 27 December 2005
Published online 7 August 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/bit.20871
Abstract: Based on the transcriptional profiling of CHO
cell culture using microarray, four key early apoptosis
signaling genes, Fadd, Faim, Alg-2, and Requiem, were
identified and CHO GT (Gene Targeted) cell lines were
developed by targeting these four genes Two were CHO
GTOcell lines overexpressing anti-apoptotic genes, Faim
and Fadd DN and two were CHO GTKDcell lines involving
knockdown of Alg-2 and Requiem which are pro-apoptotic
genes using small interfering RNA (siRNA) technology
Comparisons of these CHO GT cell lines with the parental
cell line in batch culture (BC) and fed-batch culture (FBC)
were performed Compared to parental cells, the CHO GT
cell lines showed apoptosis resistance as they
signifi-cantly delayed and/or suppressed initiator caspase-8
and -9 and executioner caspase-3 activities during culture
FBC of CHO GT cell lines reached significantly higher
maximum viable cell densities (up to 9 106
cells/mL) compared with the parental cell line (5 106
cells/mL) The recombinant interferon gamma (IFN-g) yields were
increased by up to 2.5-fold Furthermore, it was observed
that the IFN-g was more highly sialylated
ß 2006 Wiley Periodicals, Inc.
Keywords: CHO; apoptosis; fed-batch; FAIM; FADD;
ALG-2; REQUIEM
INTRODUCTION
In many bioprocesses involving animal cells, cell death is a
major barrier to maintaining high cell densities at high
viability and often leads to lower protein yields and quality
(Arden and Betenbaugh, 2004; Fussenegger and Bailey,
1998; Laken and Leonard, 2001; Vives et al., 2003a) The
ability to extend culture life at high viability is important in
processes to maintain recombinant protein quality For
example, degradative enzymes released during cell death
can detrimentally affect the sialylation of the recombinant
protein resulting in reduced circulatory half-life of
biother-apeutics in vivo (Gramer et al., 1994; Varki, 1993)
Cell death in culture has been attributed to apoptosis (Goswami et al., 1999; Singh et al., 1994) Various strategies such as nutrient feeding, the addition of anti-apoptotic chemicals, and genetic manipulation have been used to extend culture viability (Arden and Betenbaugh, 2004; Fussenegger and Bailey, 1998; Laken and Leonard, 2001; Vives et al., 2003a) The most common genetic modification reported in literature involves overexpression of either Bcl-2
or Bcl-xLto prevent mitochondrial release of cytochrome c (Arden and Betenbaugh, 2004; Laken and Leonard, 2001; Vives et al., 2003a) Alternatively, the viral homologues of Bcl-2 such as Bhrf-1 and E1B19K can also be used to confer apoptosis resistance Other strategies include approaches that interfere with caspase activation such as anti-sense RNA against caspases or overexpression of caspase inhibitors such
as XIAP and CrmA (Kim and Lee, 2002; Sauerwald et al.,
2002, 2003)
In an earlier study (Wong et al., 2006), the transcriptome analysis of apoptosis signaling pathways using microarray showed differential expression of FasL, Fadd, Bim, and Bak
in batch culture (BC) and Fadd, Faim, Bim, Bad, Bax, Alg-2 and Requiem in fed-batch culture (FBC) These were considered to be early (initiator) apoptosis signaling genes
as they were up- or downregulated going from the exponential to the stationary phase of BC and FBC As shown in Figure 1, these genes are involved in death receptor-, mitochondria-, and endoplasmic reticulum (ER)-mediated apoptosis signaling
In the extrinsic death receptor-mediated apoptosis signal-ing pathway, FasL and Fadd, which are involved in CD95 death receptor apoptosis signaling (Curtin and Cotter, 2003; Peter et al., 1996), were found to be upregulated during BC and FBC (Wong et al., 2006) We also observed significant downregulation of Faim, a pro-survival gene that confers resistance to CD95 death receptor-mediated apoptosis signaling (Rothstein et al., 2000; Schneider et al., 1999),
Correspondence to: M.G.S Yap
Trang 2suggesting that this particular survival pathway was
compromised during FBC
In the intrinsic mitochondrial-mediated pathway, we
found significant upregulation of several pro-apoptotic
BCL-2 family members such as Bak, Bax, Bim, and Bad
(Wong et al., 2006) It has been reported that the
pro-apoptotic activities of these four genes can be neutralized by
the protective properties of Bcl-2 and Bcl-xL (Cory et al.,
2003; Gross et al., 1999; O’Connor et al., 1998; van Gurp
et al., 2003) Various researchers have shown the
effective-ness of Bcl-2 and Bcl-xL overexpression for delaying the
onset of apoptosis (Charbonneau et al., 2003; Chiang
and Sisk, 2005; Chung et al., 1998; Figueroa et al., 2004;
Mastrangelo et al., 2000; Meents et al., 2002; Simpson et al.,
1999; Tey et al., 2000; Vives et al., 2003b)
In the intrinsic ER-mediated pathway, we observed the
upregulation of Apoptosis linked gene 2 (Alg-2) during
culture (Wong et al., 2006) Although it has been shown
that ALG-2 protein forms part of the ER-stress induced
caspase-activating complex (Rao et al., 2004a,b), Alg-2 gene
has also been implicated in CD95 death receptor-mediated
apoptosis (Jung et al., 2001; Krebs and Klemenz, 2000; Vito
et al., 1996)
Requiem, which encodes for a zinc finger protein
transcription factor, has been reported to be required for
apoptosis response following survival factor withdrawal
(Gabig et al., 1994) However, the exact role of Requiem in
apoptosis signaling has not been clearly defined
Out of the nine early apoptosis signaling genes found to be
differentially expressed in BC and FBC, four of these genes,
Fadd (upregulated in BC and FBC), Faim (downregulated in FBC), Alg-2 (upregulated in FBC), and Requiem (upregu-lated in FBC), were selected for gene targeting to develop apoptosis-resistant cell lines FasL was not selected for targeting because it was upregulated only in BC and not FBC Overexpression of Bcl-2 and Bcl-xL has been reported to suppress the pro-apoptotic activities of Bim, Bak, Bax, and Bad, and as such these genes were not selected In this study, four CHO GT (gene targeted) cell lines producing recombi-nant human interferon gamma (IFN-g) were constructed and compared with the parental CHO in BC and FBC in terms of culture viability, IFN-g yields, and quality
MATERIALS AND METHODS
Construction of Plasmids
FADD Dominant Negative Expression Vector
To overexpress a dominant negative form of FADD from CHO cells, an artificial Fadd dominant negative (Fadd DN) fragment with kozak sequence was created by using a 50-PCR
TGACA TTGTA TGCGA CAATG TGGGG-30 and a 30 -PCR primer, 50-CCCGG GCTCG AGTGC CTCCC TTCCA CCAGG TCAG-30 The underlined sequence consisted of a BamHI and XhoI restriction site, respectively, while the italicized sequence consisted of an artificial kozak and start codon to facilitate ‘in frame’ expression of Fadd DN A partial Fadd sequence cloned from CHO cells and subcloned
Figure 1 Apoptosis signaling via death receptor-, mitochondria-, and ER-mediated apoptosis signaling pathways during CHO cell culture (shaded box indicates genes which are targeted in this study).
Trang 3into pCR -TOPO (Invitrogen, Carlsbad, CA), was used as
the template for PCR amplification PCR conditions were:
948C for 5 min, followed by 31 cycles of 948C for 1 min, 508C
for 1 min and 728C for 2 min and a final extension at 728C for
10 min The verified PCR product was then subcloned into
pcDNA3.1(þ) (Invitrogen) and verified again by sequencing
Verified pcDNA3.1(þ) Fadd DN was then purified using
Maxi Plasmid Purification Kit (Qiagen, Valencia, CA) and its
concentration quantified for transfection into CHO IFN-g
cells to generate CHO GTOFADD DN cell line
FAIM Expression Vector
To overexpress the anti-apoptosis protein FAIM from CHO
cells, we created a FAIM with an artificial kozak sequence
and linker regions using a 50-PCR primer, 50-GAATT
CGCCA CCATG ACAGA TCTTG TAGC-30 and a 30-PCR
primer, 50-GAATT CGTGA ACACA TTTAA TTACC A-30
The underlined sequence consisted of an EcoRI restriction
site while the italicized sequence consisted of an artificial
kozak sequence to facilitate ‘in frame’ expression of
CgFAIM A Faim cDNA fragment cloned from CHO cells
and subcloned in pCR1-TOPO1 (Invitrogen) was used as
the template The PCR conditions were: 948C for 5 min,
followed by 60 cycles of 948C for 1 min, 448C for 1 min and
728C for 2min and a final extension at 728C for 10 min The
verified PCR product was cloned into the EcoRI insertion site
of pcDNA3.1(þ) vector (Invitrogen) and verified again by
sequencing Verified pcDNA3.1(þ) Faim was then purified
using Maxi Plasmid Purification Kit (Qiagen) and its
concentration quantified for transfection into CHO IFN-g
cells to generate CHO GTOFAIM cell line
ALG-2 Suppression Vector
To achieve the silencing of pro-apoptotic ALG-2 from CHO
cells, a small interfering RNA (siRNA) vector, pSUPER.neo
(OligoEngine, Seattle, WA) was inserted with a pair of
oligonucleotide insert containing unique Alg-2 sequences
shown by underlined nucleotides The 50 oligo insert, 50
-GATCCC GTGAG CTTCA GCAAG CATTA TTCAA
GAGAT AATGC TTGCT GAAGC TCATT TTTTG
GAAA-30 was annealed to the 30 oligo insert, 50-AGCTT
TTCCA AAAAA TGAGC TTCAG CAAGC ATTAT
ligated into HindIII and BglII digested pSUPER.neo vector
(OligoEngine) Verified pSUPER.neo.ALG-2 siRNA was
then purified using Maxi Plasmid Purification Kit (Qiagen)
and its concentration quantified for transfection into CHO
IFN-g cells to generate CHO GTKDALG-2 cell line
Requiem Suppression Vector
To achieve the silencing of pro-apoptotic Requiem from CHO
cells, a siRNA vector, pSUPER.neo (OligoEngine) was
inserted with a pair of oligonucleotide insert containing
unique Requiem sequences shown by underlined nucleotides
The 5 oligo insert, 5-GATCC CGCGG ATCCT TGAAC CTGAT TTCAA GAGAA TCAGG TTCAG GATCC GCTTT TTTGG AAA-30was annealed to the 30oligo insert,
TGATT CTCTT GAAAT CAGGT TCAAG GATCC
GCGG-30and ligated into HindIII and BglII digested pSUPER.neo vector Verified pSUPER.neo.Requiem siRNA plasmid was then purified using Maxi Plasmid Purification Kit (Qiagen) and its concentration quantified for transfection into CHO IFN-g cells to generate CHO GTKDREQUIEM cell line
Real-Time PCR (RT-PCR) Total RNA was extracted using TrizolTMreagent (Invitrogen) from samples containing approximately 10 million cells and quantified using GeneQuantTM Pro RNA/DNA Calculator (Amersham Biosciences, Piscataway, NJ) RNA samples were examined on a 1% denaturing RNA gel to ensure no RNA degradation after extraction Total RNA concentration and purity were determined using a UV spectrometer (Amersham Biosciences) RT-PCR was used to ascertain the relative overexpression or suppression of gene of interest after transfection experiments RT-PCR was carried out using the ABI PRISM1 7000 Sequence Detection System using SYBR1Green PCR Master Mix (Applied Biosystem, Foster City, CA) Primers used for gene-specific real-time PCR were
as described by Wong et al (2006) The primers were designed to result in amplicon sizes ranging from 100 to 200
bp In order to generate standard curves, quantified pCR1 -TOPO1(Invitrogen) plasmids containing either Faim, Fadd, Alg-2 or Requiem cloned from CHO cells were serially diluted and used as standards for RT-PCR Duplicate runs were conducted for each sample and normalized against b-actin (U20114) expression Fold change in gene expression was calculated using the delta-delta threshold cycle (DCT) method as described by Livak and Schmittgen (2001)
Cell Line CHO IFN-g is a suspension Chinese Hamster Ovary cell line that produces recombinant human IFN-g (Scahill et al., 1983) CHO IFN-g was maintained in commercially avail-able serum-free HyQ CHO MPS media (Hyclone, Logan, UT) supplemented with 4 mM glutamine, 20 mM glucose, and 0.25 mM methotrexate (Sigma, St Louis, MO)
Creation of Stable Cell Lines Transfection was carried out using Lipofectamine reagent (Invitrogen) according to the manufacturer’s instructions Cells were grown overnight in 6-well plates with 0.5 million cells/well and transfected with approximately 1 mg of linearized plasmid per well the next day To generate stable cell lines, cells were grown for 24 h before changeover to selection media (1 mg/mL of Geneticin) The cells were maintained in selection media for 4 weeks where the untransfected cells in the selection media died within a
Trang 4week Stably integrated single cell clones were obtained by
serial dilution of cells into 96-well plates such that there was
only one cell in each well Wells were checked under a light
microscope and only those containing a single cell were
marked for expansion Single cell clones were expanded into
24-well plates followed by 6-well plates before culturing in
shake flask
Batch and Fed-Batch Bioreactor
Control Operations
Batch culture and FBC operations were performed according
to methods previously described by Wong et al (2005) The
FBC utilized an online dynamic feeding strategy where at
intervals of 1.5 h, an automated measurement of residual
glutamine concentration was taken If residual glutamine fell
below setpoint control concentrations of 0.3 mM, feeding
was effected with concentrated media to raise culture
glutamine concentration to 0.3 mM
Measurement of Cell Viability and Apoptosis
Cell viability was determined by trypan blue exclusion assay
Apoptosis was determined using an Ethidium Bromide/
Acridine Orange assay adapted from Mercille and Massie
(1994) that allowed for the classification of cells into
apoptotic or non-apoptotic populations
Proteolytic Activities of Initiator
Caspase-8, -9 and Executioner Caspase–3
In addition to morphological analysis, caspase activities were
measured as biochemical indications of apoptosis induction
using BD ApoAlertTM Caspase Assay Plates (BD
Bios-ciences Clontech, CA) according to the manufacturer’s
protocol The assay kit contained substrate specific for
caspases-3, -8, and -9, which upon cleavage by the specific
caspases can be detected using a fluorescence plate reader at
380 nm excitation and 460 nm emission Normalized caspase
activity was calculated using the following equation: Normalized caspase activity¼ Fluorescencetime x
Fluorescencetime 0
Recombinant Human IFN-g Quantification Recombinant human IFN-g concentrations in culture were determined using an enzyme-linked immunosorbent (ELISA) assay (HyCult Biotechnology, Uden, The Netherlands) Average specific productivity of IFN-g was then calculated across the exponential growth phases according to the methods previously described by Wong et al (2005)
Sialic Acid Content Assay Recombinant IFN-g was purified from samples collected at mid-exponential growth phase and at when the highest IFN-g concentrations were detected during high viability (>95%) and during low viability (70–80%) The sialic acid content of the IFN-g was then determined using a modified thiobarbi-turic acid assay as described by Wong et al (2005)
RESULTS
Creation of Gene Targeted CHO (CHO GT) Cell Lines
A summary of the anti-apoptosis strategies used to confer apoptosis resistance on CHO IFN-g cells is shown in Table I For ease of reference, these genetically modified cells were termed ‘CHO GT’ followed by subscript ‘O’ or ‘KD’ to indicate overexpression or knockdown of the candidate genes CHO GTOFAIM cells were genetically modified to constitutively overexpress recombinant FAIM protein to enhance the endogenous Faim pro-survival pathway, which was observed to be compromised during FBC (Wong et al., 2006) CHO GTO FADD DN cells were genetically
Table I Fold increase/decrease in gene expression in CHO GT and control cell lines.
Cell line
Candidate apoptosis gene
Anti-apoptosis strategy
Relative increase/decrease in expression (fold change) Faim Fadd DN Alg-2 Requiem CHO GT O Blank pool None None þ0.2 0.3 0.3 0.4 — — CHO GT O FAIM pool Faim Overexpression þ3.3 0.2 — — — CHO GT O FAIM clone 5 Faim Overexpression þ3.9 0.3 — — — CHO GT O FADD DN pool Fadd Dominant negative — þ4.3 0.3 — — CHO GT O FADD DN clone 7 Fadd Dominant negative — þ4.5 0.5 — — CHO GT KD Blank pool None None — — þ0.1 0.2 0.2 0.3 CHO GT KD ALG-2 pool Alg-2 siRNA knockdown — — 2.3 0.1 — CHO GT KD ALG-2 clone 1 Alg-2 siRNA knockdown — — 3.3 0.3 — CHO GT KD REQUIEM pool Requiem siRNA knockdown — — — 3.4 0.4 CHO GT KD REQUIEM clone 4 Requiem siRNA knockdown — — — 4.2 0.2 Relative increase or decrease in expression in various cell lines compared to the parental CHO IFN-g cell line as determined by quantitative real-time PCR using primers specific for the genes examined.
Trang 5engineered to overexpress a dominant negative form of
FADD protein It has been shown that overexpression of the
dominant negative form, which contains the death domain
(DD) but not the death effector domain (DED) of FADD
allowed for effective suppression of apoptosis signaling
via FADD and CD95 death receptors (Chinnaiyan et al.,
1996)
Unfortunately, as the functional domains of the other
pro-apoptotic genes, Alg-2 and Requiem, are not well defined, it is
difficult to design a dominant negative approach Thus, a
vector that allowed for stable expression of siRNA was used
to knockdown the expression of these two pro-apoptotic
genes The siRNA can achieve gene-specific knockdown by
guiding an RNA-induced silencing complex to cleave
specific mRNAs based on sequence complement (Hammond
et al., 2001; Moss and Taylor, 2003; Samuel, 2004) The
gene-specific siRNA was designed according to criteria
described by Brummelkamp et al (2002) To further ensure
specific gene targeting, oligo insert sequences were
com-pared against Genebank database using BLAST to ensure the
sequence specificity to the desired target gene
In order to validate the effectiveness of the
above-mentioned strategies to overexpress or suppress specifically
targeted genes, we used real-time PCR to quantify specific
gene expression of either Faim, Fadd DN, Alg-2 or Requiem
in CHO GT cells Real-time PCR analysis can be used reliably for detection of mRNA abundance and has a wide dynamic range of quantification (Klein, 2002) The quanti-tative real-time PCR results showed that the CHO GT cells indeed have overexpression/suppression of the intended gene targets while the negative controls consisting of CHO GTO blank cells transfected with pcDNA3.1(þ) blank vector and CHO GTKDblank cells transfected with pSUPER.neo blank vector, did not show significant changes in gene expression when compared to the parental CHO IFN-g cells (Table I)
Batch Cultures of CHO GT Transfected Pools Figures 2 and 3 show the profiles of viable cell density, % viability and % apoptotic cells for the four different CHO GT cell lines during BC using transfected pools The use of transfected pools in this initial characterization ensured that any observed apoptosis-resistant phenotype is not due to clonal variation caused by single cell selection CHO GTO FADD DN and CHO GTOFAIM cells were able to achieve similar maximum viable cell densities comparable to the control cells (Fig 2A) The loss in culture viability for CHO
GTOFADD DN and CHO GTOFAIM cells were delayed for
Figure 2 CHO GT O cells in batch cultures The viable cell density (A), viability (B) and percentage of apoptotic cells (C) of CHO GT O FADD DN pool ( ) and CHO GT O FAIM pool ( ) compared to CHO GT O Blank pool (&) in batch cultures.
Trang 624 h (Fig 2B) corresponding to a 24 h delay in the onset of
apoptosis (Fig 2C) compared to control CHO GTO Blank
cells In the case of CHO GTKDALG-2 and CHO GTKD
REQUIEM cells, maximum viable cell densities achieved
were higher than the control CHO GTKDBlank cells (Fig 3A)
Both CHO GTKDALG-2 and CHO GTKDREQUIEM cells
also showed a 24 h delay in the loss of culture viability
(Fig 3B) and the onset of apoptosis (Fig 3C) compared to
CHO GTKDBlank cells These results indicate clearly that all
four gene-targeting strategies were able to delay the onset of
apoptosis resulting in prolonged BC
As shown in Figure 4, the extended culture viabilities (from
72 to 96 h of culture time) for CHO GTOand CHO GTKDcell
lines translated into greatly enhanced recombinant IFN-g
yields The CHO GTOand CHO GTKDcells achieved two- to
threefold higher recombinant IFN-g yields (4–6 mg/L)
compared to the average 2 mg/L yield in CHO IFN-g parental
and CHO GT Blank cells in BC Specific productivities of the
CHO GT were similar to the parental CHO IFN-g cell line as
shown in Figure 5, indicating that the improvement in
recombinant IFN-g yields achieved was due to prolonged
culture viability rather than to improvement in specific
productivities
Proteolytic Activities of Caspases in Batch Cultures
Figures 6 and 7 show the impact of early apoptosis gene targeting in CHO GTO and CHO GTKD cells on initiator caspase-8 and -9 and executioner caspase-3 activation during
BC Executioner caspase-3 is involved in the execution of apoptosis and can be activated either by initiator caspase-8, which is primarily involved in death receptor-mediated apoptosis signaling or by initiator caspase-9, which is primarily involved in mitochondria-mediated apoptosis signaling (Riedl and Shi, 2004)
For both CHO GTOFADD DN and CHO GTOFAIM cells, the onset of initiator caspase-8 activities (Fig 6A) was delayed by 72 h, while initiator caspase-9 activities (Fig 6B) were suppressed compared to the control CHO
GTOBlank cells There was a delay of 24 h in the downstream activation of executioner caspase-3 (Fig 6C) for CHO GTO FADD DN and CHO GTOFAIM cells compared with control CHO GTOBlank
In CHO GTKDALG-2 cells, low activities of caspase-8 (Fig 7A) and caspase-9 (Fig 7B) were observed, indicating minimal activation of these initiator caspases compared to
Figure 3 CHO GT KD cells in batch cultures The viable cell density (A), viability (B) and percentage of apoptotic cells (C) of CHO GT KD ALG-2 pool (~) and CHO GT KD REQUIEM pool (*) compared to CHO GT KD Blank pool (&) in batch cultures.
Trang 7CHO GTKDBlank cells However, as shown in Figure 7C, the
executioner caspase-3 was observed to be activated at 144 h,
albeit at a suppressed level with a 24 h delay compared with the
activation of executioner caspase-3 in CHO GTKDBlank cells
In the case of CHO GTKD REQUIEM cells, caspase activation profiles for caspase-8 (Fig 7A) and caspase-9 (Fig 7B) were similar to that for CHO GTOFADD DN and CHO GTOFAIM There was also a corresponding 24 h delay
in downstream activation of executioner caspase-3 (Fig 7C) compared to the control CHO GTKDBlank cells
The above results showing that the CHO GT cells exhibit significant delays and/or suppression of caspase activities compared to CHO GT Blank cells are evidences that clearly demonstrate that all four gene-targeting strategies were effective in delaying apoptosis in BC
Fed-Batch Cultures of CHO GT Cell Lines The fed-batch studies of CHO GT cells were carried out using single cell clones selected to have comparable targeted gene overexpression or suppression with the transfected pool based on real-time PCR as shown in Table I Using single cell clones as opposed to transfected pools for the fed-batch studies is advantageous as a homogeneous population can be maintained over the extended culture time This would ensure that the effects of the targeted genes (namely Fadd, Faim, Alg-2, and Requiem) on apoptosis in culture would be clearly exhibited
As shown in Figure 8, all four CHO GT cells could delay loss in culture viability by 24–48 h compared to the parental CHO IFN-g cells during FBC In addition, three of the CHO
GT cells, namely CHO GTOFAIM, CHO GTKDALG-2, and CHO GTKD REQUIEM achieved much higher viable cell densities of up to 9.0 106cells/mL compared to the parental cell line (5.0 106cells/mL) in FBC
Most importantly, Figure 9 shows that the extension in culture viability and improvement in culture cell density for CHO GT cell lines in FBC translated into improvement in recombinant protein yield Compared to the 20 mg/L of recombinant human IFN-g yield in CHO parental FBC, the best performing cell line, CHO GTKDREQUIEM showed a 2.5-fold increase in yield (up to 49 mg/L) of IFN-g during FBC
Sialylation of Recombinant Human IFN-g in CHO GT Cell Lines
Figure 10 shows the sialylation of recombinant human IFN-g harvested at three time points during FBC, namely at the mid-exponential (>95% viability), stationary (>95% viability), and death phase (70–80% viability) for the CHO GT and parental CHO IFN-g cell lines For the latter, the sialic acid content of recombinant human IFN-g decreased as the culture progressed from mid-exponential (2.9 mol of SA/mol of IFN-g) to stationary (2.3 mol of SA/mol of IFN-g) to death phase (2.1 mol of SA/mol of IFN-g) In contrast, the sialic acid content of IFN-g harvested at the three time points for the four CHO GT cell lines was maintained, and even showed increase in sialylation, ranging from 2.7 to 3.5 mol of SA/mol
of IFN-g These results seem to suggest that another potential benefit of apoptosis-resistant CHO cells is the maintenance/
Figure 4 Recombinant human IFN-g yields in batch cultures
Concentra-tions of recombinant human IFN-g during batch cultures of CHO GT O FADD
DN pool ( ), CHO GT O FAIM pool ( ), CHO GT KD ALG-2 pool (~), and
CHO GT KD REQUIEM pool (*) compared to CHO GT blank pool (&).
Figure 5 Specific productivities of recombinant human IFN-g in CHO GT
cells The four CHO GT cells, CHO GT O FADD DN pool ( ), CHO GT O FAIM
pool ( ), CHO GT KD ALG-2 pool (~), and CHO GT KD REQUIEM pool (*)
showed similar productivities to the parental CHO IFN-g cell line (&).
Trang 8enhancement of protein glycosylation quality over extended
culture time, regardless of loss in culture viability (70–80%)
DISCUSSION
Targeting of Fadd, Faim, Alg-2, and Requiem to
Prolong BC and FBC
Overcoming apoptosis, the major mode of cell death in many
bioprocesses is desirable to enhance product yield and
quality We have previously demonstrated that DNA
micro-arrays could be used to profile apoptosis signaling pathways
in culture and identified early apoptosis signaling genes
(Wong et al., 2006) In this study, we selected Fadd, Faim,
Alg-2, and Requiem to target prolonged culture viability And
as hypothesized, the results in this study showed that the four
CHO GT cell lines constructed by targeting the early
apoptosis signaling genes prolonged culture viability by
delaying the onset of apoptosis in BC and FBC Recombinant
IFN-g yields of between 1.2- to 2.5-fold higher were achieved
compared to parental CHO IFN-g cells These enhanced yields
are comparable, or in some cases better than those reported
involving either Bcl-2 or Bcl-xLoverexpression (Fussenegger
et al., 1998; Chiang and Sisk, 2005; Figueroa et al., 2004;
Jung et al., 2002; Meents et al., 2002; Tey et al., 2000)
For all four CHO GT cell lines, the enhancement in IFN-g
yields was accompanied by the notable finding that the sialic
acid content of the IFN-g produced in FBC was maintained/ enhanced and did not decrease over the extended culture time, regardless of loss in culture viability (70–80%) This is
a distinct advantage for cell lines used for manufacturing biotherapeutics as a lower degree of sialylation can decrease the in vivo half-life of protein-based drugs (Gramer et al., 1995; Varki, 1993) It is hypothesized that apoptosis-related signaling can detrimentally affect protein glycosylation As such, in CHO GT cells where the onset of apoptosis is delayed, the glycosylation machinery continues to be active, resulting in more highly sialylated glycoproteins Decrease
in sialidase released by CHO GT cells may account for the observed maintenance of sialylation of IFN-g over extended culture time and loss in culture viability
Strategies to Enhance Apoptosis Resistance Most reported anti-apoptosis strategies block apoptosis via targeting the late execution pathways such as caspase-3 activation or cytochrome c release (Arden and Betenbaugh, 2004; Laken and Leonard, 2001; Vives et al., 2003a) The advantage is that apoptosis signaling pathways converging
on a few executioner genes can be blocked simultaneously However, targeting of late executioner genes may not be as effective, as the activation of the early apoptotic signals may cause detrimental effects on cell metabolism and productivity
Figure 6 Caspase-8, -9, and -3 activities during batch culture of CHO GT O cell lines The activities of initiator caspase-8 (A), initiator caspase-9 (B), and executioner caspase-3 (C) of CHO GT O FADD DN pool ( ) and CHO GT O FAIM pool ( ) compared to CHO GT O Blank pool (&) during batch cultures (caspase activities are normalized to caspase activity at time 0 h).
Trang 9Figure 7 Caspase-8, -9, and -3 activities during batch culture of CHO GT KD cell lines The activities of initiator caspase-8 (A), initiator caspase-9 (B), and executioner caspase-3 (C) of CHO GT KD ALG-2 pool (~) and CHO GT KD REQUIEM pool (*) compared to CHO GT KD Blank pool (&) during batch cultures (caspase activities are normalized to caspase activity at time 0 h).
Figure 8 Viable cell densities of CHO GT cell lines in fed-batch cultures Viable cell densities of CHO GT O FADD DN cell line ( ), CHO GT O FAIM cell line ( ), CHO GT KD ALG-2 cell line (~) and CHO GT KD REQUIEM cell line (*) compared to the parental CHO IFN-g cell line (&) during fed-batch culture.
Trang 10In contrast, the anti-apoptosis strategies described in this
study involve targeting early apoptosis signaling genes,
acting prior to activation of caspases (Chinnaiyan et al., 1996;
Gabig et al., 1994; Jung et al., 2001; Rothstein et al., 2000;
Schneider et al., 1999) The results here showed that
intervention at the early initiation stages could delay the
onset of apoptosis, leading to enhanced culture viability and
recombinant protein yields However, the possible drawback
of targeting early initiation signals is that the strategy may be cell type- and/or stimulus-specific and the activation of other initiator apoptosis signaling pathways may compromise apoptosis resistance
A multi-gene approach which targets both early apoptosis signaling genes such as Fadd, Faim, Alg-2, and Requiem together with late executioner genes such as Caspase-3 may offer further improvement in extension of culture viability
Figure 9 Enhanced recombinant human IFN-g yields in CHO GT cell lines during fed-batch cultures Recombinant IFN-g yields for CHO GT O FADD DN cell line ( ), CHO GT O FAIM cell line ( ), CHO GT KD ALG-2 cell line (~), and CHO GT KD REQUIEM cell line (*) during fed-batch culture compared to the parental CHO IFN-g cell line during batch (&) and fed-batch (&) culture.
Figure 10 Sialylation of recombinant IFN-g in CHO GT cell lines during mid-exponential, stationary, and death phase of FBC Sialylation of IFN-g during fed-batch culture of CHO GT O FADD DN cell line ( ), CHO GT O FAIM cell line ( ), CHO GT KD ALG-2 cell line (~) and CHO GT KD REQUIEM cell line (*) compared to the parental CHO IFN-g cell line (&).