Targeting Early Apoptotic Genes in Batch and Fed-Batch CHO Cell Cultures Danny Chee Furng Wong,1,2 Kathy Tin Kam Wong,1 Peter Morin Nissom,1 Chew Kiat Heng,2 Miranda Gek Sim Yap1,3 Bioprocessing 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 Department of Paediatrics, National University of Singapore, Singapore Department of Chemical and Biomolecular Engineering, National University of Singapore, Singapore Received June 2005; accepted 27 December 2005 Published online 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 GTO cell lines overexpressing anti-apoptotic genes, Faim and Fadd DN and two were CHO GTKD cell 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 significantly 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  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 biotherapeutics in vivo (Gramer et al., 1994; Varki, 1993). Correspondence to: M.G.S. Yap ß 2006 Wiley Periodicals, Inc. 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-xL to 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 signaling 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), 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). suggesting 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 proapoptotic 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 effectiveness 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 (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 (upregulated 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 recombinant 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 primer, 50 -GATAT CGGATCC GCCA CCATG GCCTT 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 Wong et al.: Targeting Early Apoptotic Genes Biotechnology and Bioengineering. DOI 10.1002/bit 351 into pCR1-TOPO1 (Invitrogen, Carlsbad, CA), was used as the template for PCR amplification. PCR conditions were: 948C for min, followed by 31 cycles of 948C for min, 508C for and 728C for 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 GTO FADD DN cell line. The 50 oligo insert, 50 -GATCC CGCGG ATCCT TGAAC CTGAT TTCAA GAGAA TCAGG TTCAG GATCC GCTTT TTTGG AAA-30 was annealed to the 30 oligo insert, 50 -AGCTT TTCCA AAAAA GCGGA TCCTT GAACC TGATT CTCTT GAAAT CAGGT TCAAG GATCC GCGG30 and 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 GTKD REQUIEM cell line. FAIM Expression Vector Real-Time PCR (RT-PCR) 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 min, followed by 60 cycles of 948C for min, 448C for 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 GTO FAIM cell line. Total RNAwas extracted using TrizolTM reagent (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 SYBR1 Green 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 pCR1TOPO1 (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 bactin (U20114) expression. Fold change in gene expression was calculated using the delta-delta threshold cycle (DCT) method as described by Livak and Schmittgen (2001). 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 CTCTT GAATA ATGCT TGCTG AAGCT CACG-30 and 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 GTKD ALG-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. 352 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 available serum-free HyQ CHO MPS media (Hyclone, Logan, UT) supplemented with 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 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 weeks where the untransfected cells in the selection media died within a Biotechnology and Bioengineering, Vol. 95, No. 3, October 20, 2006 DOI 10.1002/bit week. 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 Biosciences 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 activity ¼ Fluorescencetime x À Fluorescencetime 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 thiobarbituric 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 GTO FAIM 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. Relative increase/decrease in expression (fold change) Cell line CHO GTO Blank pool CHO GTO FAIM pool CHO GTO FAIM clone CHO GTO FADD DN pool CHO GTO FADD DN clone CHO GTKD Blank pool CHO GTKD ALG-2 pool CHO GTKD ALG-2 clone CHO GTKD REQUIEM pool CHO GTKD REQUIEM clone Candidate apoptosis gene Anti-apoptosis strategy Faim Fadd DN Alg-2 Requiem None Faim Faim Fadd Fadd None Alg-2 Alg-2 Requiem Requiem None Overexpression Overexpression Dominant negative Dominant negative None siRNA knockdown siRNA knockdown siRNA knockdown siRNA knockdown þ0.2 Æ 0.3 þ3.3 Æ 0.2 þ3.9 Æ 0.3 — — — — — — — À0.3 Æ 0.4 — — þ4.3 Æ 0.3 þ4.5 Æ 0.5 — — — — — — — — — — þ0.1 Æ 0.2 À2.3 Æ 0.1 À3.3 Æ 0.3 — — — — — — — À0.2 Æ 0.3 — — À3.4 Æ 0.4 À4.2 Æ 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. Wong et al.: Targeting Early Apoptotic Genes Biotechnology and Bioengineering. DOI 10.1002/bit 353 engineered 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 proapoptotic 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 compared against Genebank database using BLAST to ensure the sequence specificity to the desired target gene. In order to validate the effectiveness of the abovementioned 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 quantitative 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 GTKD blank 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 and 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 GTO FAIM cells were able to achieve similar maximum viable cell densities comparable to the control cells (Fig. 2A). The loss in culture viability for CHO GTO FADD DN and CHO GTO FAIM cells were delayed for Figure 2. CHO GTO cells in batch cultures. The viable cell density (A), viability (B) and percentage of apoptotic cells (C) of CHO GTO FADD DN pool ( ) and CHO GTO FAIM pool ( ) compared to CHO GTO Blank pool (&) in batch cultures. 354 Biotechnology and Bioengineering, Vol. 95, No. 3, October 20, 2006 DOI 10.1002/bit Figure 3. CHO GTKD cells in batch cultures. The viable cell density (A), viability (B) and percentage of apoptotic cells (C) of CHO GTKD ALG-2 pool (~) and CHO GTKD REQUIEM pool (*) compared to CHO GTKD Blank pool (&) in batch cultures. 24 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 GTKD ALG-2 and CHO GTKD REQUIEM cells, maximum viable cell densities achieved were higher than the control CHO GTKD Blank cells (Fig. 3A). Both CHO GTKD ALG-2 and CHO GTKD REQUIEM 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 GTKD Blank 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 GTO and CHO GTKD cell lines translated into greatly enhanced recombinant IFN-g yields. The CHO GTO and CHO GTKD cells achieved two- to threefold higher recombinant IFN-g yields (4–6 mg/L) compared to the average 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 and 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 GTO FADD DN and CHO GTO FAIM 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 GTO Blank 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 GTO FAIM cells compared with control CHO GTO Blank. In CHO GTKD ALG-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 Wong et al.: Targeting Early Apoptotic Genes Biotechnology and Bioengineering. DOI 10.1002/bit 355 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 GTO FADD DN and CHO GTO FAIM. There was also a corresponding 24 h delay in downstream activation of executioner caspase-3 (Fig. 7C) compared to the control CHO GTKD Blank 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 Figure 4. Recombinant human IFN-g yields in batch cultures. Concentrations of recombinant human IFN-g during batch cultures of CHO GTO FADD DN pool ( ), CHO GTO FAIM pool ( ), CHO GTKD ALG-2 pool (~), and CHO GTKD REQUIEM pool (*) compared to CHO GT blank pool (&). CHO GTKD Blank 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 GTKD Blank cells. 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 GTO FAIM, CHO GTKD ALG-2, and CHO GTKD REQUIEM achieved much higher viable cell densities of up to 9.0  106 cells/mL compared to the parental cell line ($5.0  106 cells/mL) in FBC. Most importantly, Figure 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 GTKD REQUIEM 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 5. Specific productivities of recombinant human IFN-g in CHO GT cells. The four CHO GT cells, CHO GTO FADD DN pool ( ), CHO GTO FAIM pool ( ), CHO GTKD ALG-2 pool (~), and CHO GTKD REQUIEM pool (*) showed similar productivities to the parental CHO IFN-g cell line (&). 356 Figure 10 shows the sialylation of recombinant human IFN-g harvested at three time points during FBC, namely at the midexponential (>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/ Biotechnology and Bioengineering, Vol. 95, No. 3, October 20, 2006 DOI 10.1002/bit Figure 6. Caspase-8, -9, and -3 activities during batch culture of CHO GTO cell lines. The activities of initiator caspase-8 (A), initiator caspase-9 (B), and executioner caspase-3 (C) of CHO GTO FADD DN pool ( ) and CHO GTO FAIM pool ( ) compared to CHO GTO Blank pool (&) during batch cultures (caspase activities are normalized to caspase activity at time h). enhancement 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 microarrays 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-xL overexpression (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. Wong et al.: Targeting Early Apoptotic Genes Biotechnology and Bioengineering. DOI 10.1002/bit 357 Figure 7. Caspase-8, -9, and -3 activities during batch culture of CHO GTKD cell lines. The activities of initiator caspase-8 (A), initiator caspase-9 (B), and executioner caspase-3 (C) of CHO GTKD ALG-2 pool (~) and CHO GTKD REQUIEM pool (*) compared to CHO GTKD Blank pool (&) during batch cultures (caspase activities are normalized to caspase activity at time h). Figure 8. Viable cell densities of CHO GT cell lines in fed-batch cultures. Viable cell densities of CHO GTO FADD DN cell line ( ), CHO GTO FAIM cell line ( ), CHO GTKD ALG-2 cell line (~) and CHO GTKD REQUIEM cell line (*) compared to the parental CHO IFN-g cell line (&) during fed-batch culture. 358 Biotechnology and Bioengineering, Vol. 95, No. 3, October 20, 2006 DOI 10.1002/bit Figure 9. Enhanced recombinant human IFN-g yields in CHO GT cell lines during fed-batch cultures. Recombinant IFN-g yields for CHO GTO FADD DN cell line ( ), CHO GTO FAIM cell line ( ), CHO GTKD ALG-2 cell line (~), and CHO GTKD REQUIEM cell line (*) during fed-batch culture compared to the parental CHO IFN-g cell line during batch (&) and fed-batch (&) culture. In 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 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 GTO FADD DN cell line ( ), CHO GTO FAIM cell line ( ), CHO GTKD ALG-2 cell line (~) and CHO GTKD REQUIEM cell line (*) compared to the parental CHO IFN-g cell line (&). Wong et al.: Targeting Early Apoptotic Genes Biotechnology and Bioengineering. DOI 10.1002/bit 359 and recombinant protein production. Alternatively, a dual gene-targeting strategy, which simultaneously blocks the death receptor- and mitochondria-mediated pathways, may also be effective. An example drawn from this study would be to combine the overexpression of Fadd DN or Faim (which targets the death receptor-mediated apoptosis signaling pathway) with the overexpression of Bcl-2 or Bcl-xL (mediated apoptosis signaling pathway). Indeed, Figueroa et al. (2004) has shown that the overexpression of Bcl-xL together with Aven, which inhibits caspase-9, was more effective for apoptosis suppression than the individual overexpression of these genes (Figueroa et al., 2004). CONCLUSION This study showed that the targeting of early apoptosis signaling genes, Fadd, Faim, Alg-2, and Requiem, identified via transcriptome analysis, are promising strategies, which can effectively suppress apoptosis in culture. Most importantly, the prolonged culture viability can be translated into improved recombinant protein yield and quality. 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DOI 10.1002/bit 361 [...]... coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bcl-xL on productivity, cell survival and mitochondria number in CHO- DG44 grown in suspension and serum-free media Biotechnol Bioeng 80:706–716 Mercille S, Massie B 1994 Induction of apoptosis in nutrient-deprived cultures of hybridoma and myeloma cells Biotechnol Bioeng 44:1140– 1154 Moss EG, Taylor JM 2003 Small-interfering RNAs in. .. strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures Biotechnol Bioeng 89:164–177 Wong CFD, Wong TKK, Lee YY, Nissom PM, Heng CK, Yap MGS 2006 Transcriptional profiling of apoptotic pathways in batch and fed- batch CHO cell cultures Biotechnol Bioeng 94:373–382 Wong et al.: Targeting Early Apoptotic Genes Biotechnology and Bioengineering DOI 10.1002/bit 361 ... analysis, are promising strategies, which can effectively suppress apoptosis in culture Most importantly, the prolonged culture viability can be translated into improved recombinant protein yield and quality NOMENCLATURE Alg-2 Apoptosis linked gene 2 Fadd Fas associated death domain gene Faim Fas apoptosis inhibitory molecule The authors acknowledge Ong Peh Fern, Jennifer Lo, Tan Kher Shing, and Breana Cham... growth rate and prolongs G1 phase in continuous chemostat cultures of hybridoma cells Biotechnol Bioeng 64: 174–186 Singh RP, Al-Rubeai M, Gregory CD, Emery AN 1994 Cell death in bioreactors: A role for apoptosis Biotechnol Bioeng 44:720–726 Tey BT, Singh RP, Piredda L, Piacentini M, Al-Rubeai M 2000 In uence of Bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a... susceptibility to Fas-mediated apoptosis and a novel Fas apoptosis inhibitory molecule Immunol Rev 176:116–133 Sauerwald TM, Betenbaugh MJ, Oyler GA 2002 Inhibiting apoptosis in mammalian cell culture using caspase inhibitor XIAP and deletion mutants Biotechnol Bioeng 77:704–716 Sauerwald TM, Oyler GA, Betenbaugh MJ 2003 Studying caspase inhibitors for limiting death in mammalian cell culture Biotechnol... 68:31–43 van Gurp M, Festjens N, van Loo G, Saelens X, Vandenabelle P 2003 Mitochondrial intermembrane proteins in cell death Biochem Biophys Res Commun 304:487–497 Varki A 1993 Biological roles of oligosaccharides: All of the theories are correct Glycobiology 3:97–130 Vito P, Lacana E, D’Adamio L 1996 Interfering with apoptosis: Ca2þ binding protein ALG-2 and Alzheimer’s disease gene ALG-3 Science 271:521–525... cell-cycle progression and apoptosis in mammalian cells: Implications for Biotechnology Biotechnol Prog 14:807–833 Gabig TG, Mantel PL, Rosli R, Crean CD 1994 Requiem: A novel zinc finger gene essential for apoptosis in myeloid cells J Biol Chem 269: 29515–29519 Goswami J, Sinskey AJ, Steller H, Stephanopoulos GN, Wang DI 1999 Apoptosis in batch cultures of Chinese hamster ovary cells Biotechnol Bioeng... HA, Leonard MW 2001 Understanding and modulating apoptosis in industrial cell culture Curr Opin Biotechnol 12:175–179 Livak KJ, Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2^[-delta delta Ct] method Methods 25:402–408 Mastrangelo AJ, Hardwick JM, Bex F, Betenbaugh MJ 2000 Part I Bcl-2 and Bcl-x(L) limit apoptosis upon infection with alphavirus... gene 2 binds to the death domain of Fas and dissociates from Fas during Fas-mediated apoptosis in Jurkat cells Biochem Biophys Res Commun 288:420–426 Jung D, Cotes S, Drouin M, Simard C, Lemieux R 2002 Inducible expression of Bcl-XL restricts apoptosis resistance to the antibody secretion phase in hybridoma cultures Biotechnol Bioeng 79: 180–187 Kim NS, Lee GM, 2002 Inhibition of sodium butyrate-induced... sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressed ansense RNA of caspase-3 Biotechnol Bioeng 78:217–228 Klein D 2002 Quantification using real-time PCR technology: Application and limitations Trends Mol Med 8:257–260 Krebs J, Klemenz R 2000 The ALG-2/AIP-complex, a modulator at the interface between cell proliferation and cell death? A hypothesis Biochim . 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. 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. time) for CHO GT O and CHO GT KD cell lines translated into greatly enhanced recombinant IFN-g yields. The CHO GT O and CHO GT KD cells achieved two- to threefold higher recombinant IFN-g yields