Enhancing recombinant protein yield and quality using novel CHO GT cells in high density fed batch cultures 1

Enhancing Recombinant Protein Yield & Quality Using Novel CHO GT Cells in High Density Fed-batch Cultures WONG CHEE FURNG A thesis submitted for the degree of Doctor of Philosophy Department of Paediatrics Faculty of Medicine National University of Singapore 2006 Enhancing Recombinant Glycoprotein Yield & Quality Using Novel CHO GT Cells in High Density Fed-batch Cultures SUMMARY Chinese Hamster Ovary (CHO) cells are regarded as one of the ‘work-horses’ for complex biotherapeutics production. Currently, batch (BC) and fed-batch (FBC) cultures are the main culture modes for a vast majority of industrial bioprocesses due to their ease of operation and reliability. During both BC and FBC, loss in viability attributed to apoptosis often results in lower recombinant protein yield and affects protein quality. It is hypothesized that extension of culture life can potentially improve recombinant glycoprotein yield and quality. Using an ‘in-house’ developed CHO cDNA array and a mouse oligonucleotide array for time profile expression analysis of CHO BC and FBC, the genetic circuitry that regulates and executes apoptosis induction were examined. Genes such as Fadd, Faim, Alg-2, and Requiem were identified to be key apoptosis signaling genes during CHO cell cultures. Four CHO GT (Gene Targeted) cell lines were developed, in which each of these early apoptotic genes was either knocked down or overexpressed. These novel cell lines were shown to be effective in prolonging culture life resulting in higher cell densities and significantly enhancing glycoprotein yield and quality. ACKNOWLEDGEMENTS Despite being listed as the sole author of this thesis, this project has had ample contributions from a lot of people, all of whom I am extremely grateful to. To my supervisors Dr. Heng Chew Kiat and Prof. Miranda Yap, thank you very much for being your patient guidance these the past years. To Dr. Heng, thanks for caring more than just project schedules and timelines and to Prof. Yap, thanks for pushing me to be more than just a ‘lab-rat’ and that there is more to science than just the lab. Special thanks too to my friends in the Animal Cell Technology group at BTC, who welcomed me into the group with open arms and had shared some exciting and challenging times in the research arena with me. Kathy for her guidance and support, Chun Loong for successfully ‘knocking’ bioreactor principles into my brain and his ‘weird’ insights, Yih Yean for his endless help and fish-tales, Vesna for keeping things in perspective, Niki for her friendship and inputs and of course not forgetting, Victor, Yan Ying, Janice, Sanny and Poh Choo. My heartfelt appreciation to BTI’s Analytics group led by Dr. Goh Lin-Tang and Dr. Lee May May for their support in glycosylation analysis. Many thanks to Sim Lyn Chiin, Ong Boon Tee and Tracy. The BTI Microarray group: Dr. Peter Morin Nissom, Jennifer Lo, Tan Kher Shing, Ong Peh Fern, Breanna Cham and Chuah Song Hui who had helped so much in getting the CHO and mouse chips up and going. Of course, there’s the help given by the undergraduate students whom worked with me for their industrial attachment. Many thanks to Andrew Wu, Wei Jan, Wong Ju Wei, Nick Lim, Zeon Na, Amanda Lanza, Ang Pei Ling, Emily Lau and Dennis Goh for their help and enthusiasm! Last but not least, my family for being so understanding and supportive throughout the years. Thanks especially to my wife, Winnie for being beside me through the good and the bad times. Many thanks to my parents for their love and nurture and allowing me to pursue my own passion. Thanks everyone! This project would not be what it is without all of you. All the research work described in this thesis was carried out in the Bioprocessing Technology Institute (BTI), funded by the Biomedical Research Council (BMRC) established under the Agency for Science, Technology and Research (A*STAR). TABLE OF CONTENTS: Summary Acknowledgements Table of Contents List of Figures 15 List of Tables 18 CHAPTER Introduction 19 1.1 Background 19 1.1 Thesis Objectives 21 1.3 Thesis Organization 21 CHAPTER Literature Review 23 2.1 Biotherapeutics Production In Mammalian Cell Culture 23 2.1.1 Batch Cultures 25 2.1.2 Fed-Batch Cultures 25 (A) Feed Media Design 26 (B) Feeding Strategy 26 2.1.3 Accumulation of Toxic Waste Metabolites 27 2.1.4 Reduction of Metabolite Waste Production 28 2.2 The Importance of Protein Glycosylation 28 2.2.1 N-Glycosylation 30 2.2.2 Heterogeneity in N-Glycosylation 30 2.2.3 Factors Affecting Glycosylation 31 (A) Host Expression System 32 (B) Culture Environment 33 (C) Extracellular Degradation 34 2.3 Cell Death In Bioreactors 34 2.3.1 Apoptosis vs. Necrosis 35 2.3.2 Triggers of Apoptosis in Bioprocesses 36 2.3.3 Caspases, the central executioners of apoptosis 37 2.3.4 Apoptosis Signaling 38 2.3.5 Suppressing Apoptosis in Culture 40 2.4 Transcriptome Analysis 41 2.4.1 Microarray Technology 41 2.4.2 Transcription Expression Profiling 42 CHAPTER 3.1 3.1.1 3.1.1 Materials & Methods 44 Cell Lines 44 CHO IFN-γ 44 CHO GTO FADD DN 44 3.1.1 CHO GTO FAIM 44 3.1.1 CHO GTKD ALG-2 44 3.1.1 CHO GTKD REQUIEM 44 3.2 Cell Culture Maintenance 45 3.2.1 Working Cell Bank 45 3.2.2 Culture Maintenance 45 3.3 Bioreactor Culture Operations 45 3.3.1 Batch Culture Operations 45 3.3.2 Fed-batch Culture Operations 46 (A) Glutamine-controlled FBC 48 (B) Glucose-controlled FBC coupled with glutamine profile feeding 48 3.4 Metabolite Analysis 49 3.4.1 Glucose, Glutamine, Glutamate and Lactate measurement 49 3.4.2 Ammonia measurement 49 3.4.3 Amino Acid Analysis 49 3.5 Recombinant Glycoprotein Yields Determination 50 3.5.1 Determining IFN-γ Yield by ELISA 50 3.5.2 Average Specific Rates Calculations 50 3.6 N-Glycosylation Quality Of IFN-γ 50 3.6.1 Immunoaffinity Purification of IFN-γ 50 3.6.2 IFN-γ Macro-heterogeneity: Site-occupancy 51 3.6.3_ IFN-γ Micro-heterogeneity: Structural Composition of Oligosaccharides 51 (A) Determination of Oligosaccharide Species using Mass Spectrometry 52 (i) Tryptic digestion and glycopeptides separation 52 (ii) Reverse phase HPLC separation of IFN-γ glycopeptides 52 (iii) Glycopeptides analysis using MALDI/TOF mass spectometry 53 (B) Quantification of Oligosaccharide Species Using High pH 53 anion-exchange chromatography (HPAEC) (i) Enzymatic release of glycans from IFN-γ 54 (ii) Preparation of Glycan Standards 54 (iii) Purification of Released Glycans 54 (iv) HPAEC Operation 55 3.6.4 Sialylation Assay 56 3.7 Cell Viability and Apoptosis Detection 57 3.7.1 Trypan Blue Exclusion Viability Assay 57 3.7.2 Morphological Detection of Apoptosis 58 3.7.3 Biochemical Detection of Apoptosis 58 3.8 Transcriptome Analysis 60 3.8.1 Total RNA Purification 61 3.8.2 Microarray Construction 61 (A) Slide coating 61 (B) Printing of DNA probes 62 (C) DNA Immobilization & Preparation of Microarray Slides 63 Microarray Hybridization 63 (A) cDNA Synthesis 63 (B) Dye-labeling of cDNA 64 (C) Microarray Hybridization and Wash 64 3.8.3 3.8.4 Microarray Image and Data Analysis 65 3.8.5 Real-Time PCR Validation 65 3.9 Cloning of Apoptotic Genes 66 3.9.1 Bacterial Culture 66 (A) Bacterial Cells 66 (B) Culture Broth and Agar Plates 66 Vectors 67 (A) pCR®-TOPO® Vector 67 (B) pcDNA3.1(+) Vector 68 (C) pSUPER.neo Vector 68 Gene Cloning 69 (A) Gene specific Polymerase Chain Reaction (PCR) 69 (B) Rapid Amplification of cDNA ends (RACE) 69 (C) DNA Restriction & Ligation 69 (D) DNA Sequencing 70 3.9.2 3.9.3 3.9.4 (i) Cycle Sequencing PCR 70 (ii) Ethanol Purification of Cycle Sequencing PCR Product 70 (iii) Electrophoresis 70 CDNA Cloning of Fadd, Faim, Alg-2 and Requiem 71 (A) Fadd 71 (B) Faim 71 (C) Alg-2 71 (D) Requiem 72 3.10 Vector Construction For the Creation of CHO GT cells 73 3.10.1 pcDNA3.1(+) cg FADD Dominant Negative 73 3.10.2 pcDNA3.1(+) cg FAIM 74 3.10.3 pSUPER.neo cg ALG-2 74 3.10.4 pSUPER.neo cg REQUIEM 75 3.11 Creation of Stable Cell Lines 75 3.11.1 Selecting for Stable Expression Transfected Pool 75 3.11.2 Selecting for Single Cell Cloning 76 3.12 Quantitative Real Time PCR 76 3.12 Determination of Statistical Significance 77 CHAPTER High Density Fed-batch Cultures of CHO Cells 79 INTRODUCTION 79 RESULTS 80 4.1 CHO Cell Growth & Metabolism in FBC 81 4.1.1 Glutamine-controlled FBC (FBC0.1, FBC0.3 and FBC0.5) 81 (A) Tight control of glutamine concentrations 81 (A) Higher viable cell density and specific growth rates 82 (A) Reduced ammonia production 84 (A) Reduced lactate production 85 Glucose-controlled FBC (FBC0.3/0.35 and FBC0.3/0.70) 87 (A) Tight control of glucose concentrations 88 (A) Viable cell density and specific growth rates 89 (A) Reduction of lactate production 89 (A) Increased glutamine consumption and ammonia production 91 4.1.2 10 Mancuso A, Sharfstein ST, Fernandez EJ, Clark DS and Blanch HW. 1998. Effect of extracellular glutamine concentration on primary and secondary metabolism of a murine hybridoma: An in vivo 13 C nuclear magnetic resonance study. Biotechnol Bioeng 57: 172-186 Mastrangelo AJ and Betenbaugh MJ. 1998. Overcoming apoptosis: new methods for improving protein-expression systems. Trends Biotechnol 16: 88-95 Mastrangelo AJ, Hardwick JM, Bex F and Betenbaugh MJ. 2000. Part I. Bcl-2 and Bclx(L) limit apoptosis upon infection with alphavirus vectors. Biotechnol Bioeng 67: 555-564 Meents H, Enenkel B, Eppenberger HM, Werner RG and Fussenegger M. 2002. Impact of coexpression and coamplification of sICAM and antiapoptosis determinants bcl-2/bclxL on Productivity, Cell survival and Mitochondria number in CHO-DG44 grown in suspension and serum-free media. Biotech Bioeng 80: 706-716 Mercille S and Massie B. 1994. Induction of apoptosis in nutrient-deprived cultures of hybridoma and myeloma cells. Biotechnol Bioeng. 44: 1140-1154 Minch SL, Kallio PT and Bailey JE. 1995. Tissue plasminogen activator coexpressed in CHO cells with α(2,6)-sialyltransferase contains NeuAcα(2,6)Galβ(1,4)Glc-N-AcR Linkages. Biotechnol Prog 11: 348-351 Monaco L, Marc A, Eon-Duval A, Acerbis G, Distefano G, Lamotte D, Engasser JM, Soria M and Jenkins N. 1996. Genetic engineering of α2,6-sialyltransferase in recombinant CHO cells and its effects on the sialylation of recombinant interferon-γ. Cytotechnology 22: 197-203 Moore SE and Spiro RG. 1990. Demonstration that Golgi endo-alpha-D-mannosidase provides a glucosidase-independent pathway for the formation of complex N-linked oligosaccharides of glycoproteins. J Biol Chem 265: 13104-12 Mori M, Uchida M, Watanabe T, Kirito K, Hatake K, Ozawa K and Komatsu N. 2003. Activation of extracellular signal-regulate kinases ERK1 and ERK2 induce Bcl-xL upregulation via inhibition of caspase activities in erythropoietin signaling. J Cell Physiol. 195: 290-297 Moss EG and Taylor JM. 2003. Small-interfering RNAs in the radar of the interferon 167 system. Nat Cell Biol. 5: 771-772 Murray K, Ang CE, Gull K, Hickman JA and Dickson AJ. 1996. NS0 myeloma cell death: influence of bcl-2 over-expression. Biotechnol Bioeng. 51: 298-304 Nyberg GB, Balcarcel RR, Follstad BD, Stephanapoulous G and Wang DIC. 1999. Metabolic effects on recombinant interferon-γ glycosylation in continuous culture of Chinese hamster ovary cells. Biotechnol Bioeng. 62: 336-347 O’Connor L, Strasser A, O’Reilly LA, Hausmann G, Adams JM, Cory S and Huang DCS. 1998. Bim: a novel member of the Bcl-2 family that promotes apoptosis. EMBO J. 17: 384-395 Omasa T, Higashiyama K, Shioya S and Suga K. 1992. Effects of lactate concentration on hybridoma culture in lactate-controlled FBC operation. Biotechnol Bioeng. 39: 556-564. Ossina NK, Cannas A, Powers VC, Fitzpatrick PA, Knight JD, Gilbert JR, Shekhtman M , Tomei DL, Unmansky SR and Kiefer MC. 1997. Interferon-γ modulates a p53independent apoptotic pathway and apoptosis-related gene expression. J Biol Chem. 272: 16351-16357 Ozturk SS and Palsson BO. 1990. Chemical decomposition of glutamine in cell culture media: Effect of media type, pH, serum concentration. Biotechnol Prog. 6: 121-128 Parekh RB. 1991. Mammalian cell gene expression: protein glycosylation. Curr Opin Biotechnol. 2: 730-4 Pels Rijcken WRP, Overdijk B, Vandeneijnden DH, Ferwerda W. 1995. The effect of increasing nucleotide sugar concentrations on the incorporation of sugars into glycoconjugates in rat hepatocytes. Biochem J. 305: 865-870 Peter ME and Krammer PH. 2003. The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ. 10: 26-35 Peter ME, Kischkel FC, Hellbardt S, Chinnaiyan AM, Krammer PH and Dixit VM. 1996. CD95(APO-1/Fas)-associating signaling proteins. Cell Death Differ. 3: 161-170. Potvin B, Kumar R, Howard DR and Stanley P. 1990. Transfection of a human α-1,3fucosyltransferase gene in to Chinese hamster ovary cells. Complications arise from activation of endogenous α-1,3-fucosyltransferases. J Biol Chem 265: 1615-1622 168 Rademacher TW, Parekh RB and Dwek RA. 1988. Glycobiology. Ann Rev Biochem 57: 785-838 Rao R, Poksay K, Castro-Obregon S, Schilling B, Row RH, Rio G, Gibson BW, Ellerby Hm and Bredesen DE. 2004a. Molecular components of a cell death pathway activated by endoplasmic reticulum stress. J Biol Chem. 279: 117-187 Rao RV, Ellerby HM and Bredesen DE. 2004. Coupling endoplasmic reticulum stress to the cell death program. Cell Death Diff. 11: 372-380. Rao RV, Hermel E, Castro-Obregon S, del Rio G, Ellerby LM, Ellerby HM, Bredesen DE. 2001. Coupling endoplasmic reticulum stress to the cell death program. Mechanism of caspase activation. J Biol Chem. 276: 33869-74. Riedl SJ and Shi Y. 2004. Molecular mechanisms of caspase regulation during apoptosis. Nat Rev Mol Cell Biol. 5: 897-907 Rothstein TL, Zhong X, Schram BR, Negm RS, Donohoe TJ, Cabral DS, Foote LC, Schneider TJ. 2000. Receptor-specific regulation of B-cell susceptibility to Fasmediated apoptosis and a novel Fas apoptosis inhibitory molecule. Immunol Rev. 176: 116-33 Rothstein TL. 2000. Inducible resistance to Fas-mediated apoptosis in B cells. Cell Res. 10: 245-266 Roy S and Nicholson DW. 2000. Cross-Talk in Cell Death Signaling. J Exp Med. 192(8): F21-26 Rubinstein S, Familetti PC and Pestka S. 1981. Convenient assay for interferons. J. Virol. 37(2): 755-758 Rudel T and Bokoch GM. 1997. Membrane and morphological changes in apoptotic cells regulated by caspases mediated activation of PAK2. Science. 276: 1571-1574 Rytll T, Valley U and Wagner R. 1994. Biochemistry of growth Inhibition by Ammonium ions in mammalian cells. Biotechnol Bioeng. 44: 184-193 Samuel CE. 1991. Antiviral actions of interferon. Interferon-regulated cellular proteins and their surprisingly selective antiviral activities. Virology. 183: 1–11. Sareneva T, Mortz E, Tolo H, Roepstorff P, Julkunen I. 1996. Biosynthesis and Nglycosylation of human interferon-gamma. Asn25 and Asn97 differ markedly in how 169 efficiently they are glycosylated and in their oligosaccharide composition. Eur J Biochem. 242: 191-200. Sareneva T, Pirhonen J, Cantell K, Julkunen I. 1995. N-glycosylation of human interferongamma: glycans at Asn-25 are critical for protease resistance. Biochem J. 308: 9-14. Sareneva T, Pirhonen J, Cantell K, Kalkkinen N, Julkunen I. 1994. Role of N-glycosylation in the synthesis, dimerization and secretion of human interferon-gamma. Biochem J. 303: 831-40. Sasaki H, Bothner B, Dell A and Fukuda M. 1987. Carbohydrate structure of erythropoietin expressed in Chinese Hamster Ovary Cells by a human erythropoietin cDNA. J Biol Chem. 262: 12059-12076 Sauerwald TM, Betenbaugh MJ, Oyler Ga. 2002. Inhibiting apoptosis in mammalian cell culture using caspase inhibitor XIAP and deletion mutants. Biotechnol Bioeng 77: 704716 Sauerwald TM, Oyler GA, Betenbaugh MJ. 2003. Studying of caspase inhibitors for limiting death in mammalian cell culture. Biotechnol Bioeng 81: 329-340 Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, Debatin KM, Krammer PH and Peter ME. 1998. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 17:1675-1687 Scaffidi C, Schmitz I, Zha.J, Korsmeyer SJ, Krammer PH, and Peter ME. 1999. Differential modulation of apoptosis sensitivity in CD95 type I and type II cells. J Biol Chem 274: 22532-22538 Scahill, SJ, Devos R, Der Heyden VJ and Fiers W. 1983. Expression and characterization of the product of a human immune interferon cDNA gene in Chinese hamster ovary cells. PNAS 80: 4654-4658 Schena M, Shalon D, Davis RW and Brown PO. 1995. Quantitative monitoring of gene expression patterns with complementary DNA microarray. Science. 270: 467-470. Schneider TJ, Fischer GM, Donohoe TJ, Colarusso TP and TL Rothstein. 1999. A Novel Gene Coding for a Fas Apoptosis Inhibitory Molecule (FAIM) Isolated from Inducibly Fas-resistant B Lymphocytes. J. Exp. Med. 189: 949-956 Scorrano L, Oakes SA, Opferman JT, Cheng EH, Sorcinelli MD and Pozzan T. 2003. 170 BAX and BAK regulation of endoplasmic reticulum Ca2+: a control point for apoptosis. Science. 300: 135-139 Sharp PA. 1999. RNAi and double-stranded RNA. Genes Dev. 13: 139-141 Shin S, Sung BK, Cho YS, Kim HJ, Ha NC, Hwang JI, Chung CW, Jung YK and Oh BH. 2001. An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and –7. Biochemistry. 40: 1117-1123 Simonsen CC and McGrogan M. 1994. The molecular biology of production cell lines. Biologicals. 22: 1219-1224 Simpson NH, Singh RP, Emery AN and Al-Rubeai M. 1999. Bcl-2 overexpression reduced growth rate and prolongs G1 phase in continuous chemostat cultures of hybridoma cells. Biotechnol Bioeng. 64: 174-186 Simpson NH, Singh RP, Perani A, Goldenzon C, Al-Rubeai M. 1998. In hybridoma cultures, deprivation of any single amino acid leads to apoptotic death, which is suppressed by the expression of the bcl-2 gene. Biotechnol Bioeng. 59: 90-98 Singh RP, Al-Rubeai M, Gregory CD, Emery AN. 1994. Cell death in bioreactors: a role for apoptosis. Biotechnol Bioeng. 44: 720-726 Spanaus KS, Schlapback R, Fontana A. 1998. TNF-alpha and IFN-gamma render microglia sensitive to Fas ligand-induced apoptosis by inducing Fas expression and downregulation of Bcl-2 annd Bcl-xL. Eur J Immunol 28: 4398-4408 Strichman R, Samuel CE. 2001. The role of gamma interferon in antimicrobial immunity. Curr Opin Microbiol. 4: 251-9. Sun Y. 1999. Alterations of SAG mRNA in human cancer cell lines: requirement for the RING finger domain for apoptosis protection. Carcinogenesis. 20: 1899-903. Sunstrom NA, Gay RD, Wong DCF, Kitchen NA, De Boer L and Gray PP. 2000. Insulinlike growth factor-I and transferrin mediate growth and survival of Chinese hamster ovary cells. Biotechnol Prog. 16: 698-702 Suzuki N and Lee YC. 2004. Site-specific N-glycosylation of chicken serum IgG. Glycobiology. 14: 275-292 Szegezdi E, Fitzgerald U and Samali A. 2003. Caspase-12 and ER-stress-mediated apoptosis. Ann NY Acad Sci. 1010: 186-194. 171 Tekautz TM, Zhu K, Grenet J, Kaushal D, Kidd VJ, Lahti JM. 2006. Evaluation of IFNgamma effects on apoptosis and gene expression in neuroblastoma- Preclinical studies. Biochim Biophys Acta 1763(10): 1000-1010 Tey BT, Singh RP, Piredda L, Piacentini M and Al-Rubeai M. 2000. Influence of Bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody. Biotech Bioeng. 68: 31-43 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25: 4876-4882. Thurl S, Muller-Werner B and Sawatzki G. 1996. Quantification of individual oligosaccharide compounds from human milk using high-pH anion-exchange chromatography. Anal Biochem. 235: 202-206 Ting AT, Pimentel-Muinos FX and Seed B. 1996. RIP mediates tumor necrosis factor receptor activation of NF-κB but not Fas/APO-1-initiated apoptosis. EMBO J. 15: 6189-6196 Urlaub and Chasin. 1980. Isolation of Chinese hamster cell mutants deficient in dihydrofolate reductase activity Proc Natl Acad Sci USA. 77: 4216-4220 van Gurp M, Festjens N, van Loo G, Saelens X and Vandenabelle P. 2003. Mitochondrial intermembrane proteins in cell death. Biochem Biophys Res Comm. 304: 487-497 Varki A. 1993. Biological roles of oligosaccharides: All of the theories are correct. Glycobiology. 3: 97-130 Vassilev A, Ozer Z, Navara C, Mahajan S and Uckun FM. 1999. Bruton’s tyrosine kinase as an inhibitor of the Fas/CD95 death-inducing signaling complex. J Biol Chem. 274: 1646-1656 Vito P, Lacana E and D’Adamio L. 1996. Interfering with apoptosis: Ca2+ binding protein ALG-2 and Alzheimer’s disease gene ALG-3. Science. 271: 521-525 Vives J, Juanola S, Cairo JJ and Godia F. 2003a. Metabolic engineering of apoptosis in cultured animal cells: implications for the biotechnology industry. Biotechnol Prog. 5: 124-132 172 Vives J, Juanola S, Cairo JJ, Prats E, Cornudella L, Godia F. 2003b. Protective effect of viral homologues of Bcl-2 on hybridoma cells under apoptosis-inducing conditions. Biotechnol Prog 19: 84-89 Walsh G. 2003. Biopharmaceutical benchmarks- 2003. Nat Biotechnol. 21: 865-870 Wang MD, Yang M, Huzel N, Butler M. 2002. Erythropoietin production from CHO cells grown by continuous culture in a fluidized-bed bioreactor. Biotechnol Bioeng. 77: 194203 Wang SH, Mezosi E, Wolf JM, Cao Z, Utsugi S, Gauger PG, Doherty GM and Baker JJR. 2004. IFN-γ sensitization to TRAIL-induced apoptosis in human thyroid carcinoma cells by upregulating Bak expression. Oncogene. 23: 928-935 Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB, Korsmeyer SJ. 2001. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science. 292: 727-730 Wlaschin KF, Nissom PM, Gatti ML, Ong PF, Arleen S, Tan KS, Rink A, Cham B, Wong TKK, Yap MGS and Hu WS. 2005. EST Sequencing for gene discovery in Chinese Hamster Ovary cells. Biotechnol Bioeng 91: 592-606 Wong CFD, Wong TKK, Goh LT, Heng CK and Yap GSM. 2005a. Impact of dynamic online FBC 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 and Yap GSM. 2005b. Elucidating Apoptosis pathways in CHO cell cultures through transcriptome analysis. Submitted to Biotechnol Bioeng in October 2005 Wong CFD, Wong TKK, Nissom PM, Heng CK and Yap GSM. 2005c. Rational metabolic engineering of apoptosis-resistant CHO cell lines. Submitted to Biotechnol Bioeng in October 2005. Wong CFD. 1998. Apoptosis & Recombinant Protein Production in CHO Cells. B.Sc (Hons) Thesis, Dept Biotechnol, UNSW, Australia. Wurm FM. 2004. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol. 22: 1393-1398 173 Xie L, Nyberg G, Gu X, Li H, Mollborn F, Wang DIC. 1997. Gamma-Interferon Production and Quality in Stoichiometric FBC of Chinese Hamster Ovary Cells under Serum-free Conditions. Biotechnol Bioeng. 56: 577-582 Yang E, Zha J, Jockel J, Boise LH, Thompson CB, Korsmeyer SJ. 1995. Bad, a heterogimeric partner for Bcl-X(L) and Bcl-2, displaces Bax and promotes cell death. Cell 80: 285-291 Yang M, Butler M. 2000. Effects of Ammonia on CHO Cell Growth, Erythropoietin Production and Glycosylation. Biotechnol Bioeng 68: 370-380 Yang YH, Duboit S, Luu P, Lin DM, Peng V, Ngai J and Speed T. 2002. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Research 30: 1-10 Yuan J. 1997. Transducing signals of life and death. Curr Opin Cell Biol. 9: 247-251 Yuk IH, Wang DI. 2002. Changes in overall extent of protein glycosylation by Chinese hamster ovary cells over the course of BC culture. Biotechnol Appl Biochem. 36: 133140 Zhang H, Holzgreve W, De Geyter C. 2001. Bcl2-L-10, a novel anti-apoptotic member of the Bcl-2 family, blocks apoptosis in the mitochondria death pathway but not in the death receptor pathway. Hum Mol Genet 10: 2329-39. Zhou W and Hu WS. 1994. Online characterization of a hybridoma cell culture process. Biotechnol Bioeng. 44: 170-177 Zhou W, Rehm J, Hu WS. 1995. High viable cell concentration fed-batch culture of hybridoma cells through online nutrient feeding. Biotechnol Bioeng. 46: 579-587 174 APPENDIX (A) The effects of ex vivo addition of Ammonia on the viability and growth rates of CHO cells. No reduction in culture viability was observed at up to 12mM of Ammonia. Ammonia Effects on Viability 100 Ammonia Effects on Growth Rates 0.035 Growth Rates (hr ) 96 -1 Average Viability (%) 98 94 92 90 88 86 84 0.030 0.025 0.020 0.015 82 0.010 80 10 12 14 10 12 14 Ammonia (mM) Ammonia (mM) (B) The effects of ex vivo addition of Lactate on the viability and growth rates of CHO cells. Culture viability was reduced at 100mM or more of Lactate. Lactate Effects on Viability 100 Lactate Effects on Growth Rates 0.040 96 Growth Rates (hr-1) Average Viability (%) 98 94 92 90 88 86 84 0.035 0.030 0.025 0.020 0.015 82 0.010 80 10 20 30 40 50 60 70 80 90 100 110 120 130 Lactate (mM) 10 20 30 40 50 60 70 80 90 100 110 120 130 Lactate (mM) 175 APPENDIX Fas Associated Death Domain (Fadd) 61 121 181 241 301 361 421 481 541 CCATGGACCC TGCTGGAGCT AGAGTGGCCT CCGGGCTGCT ACGACTTTGA CCTTTGACAT AAGTGTCTGA AGGTAAGGGA GACTGGTAAA GG ATTCCTGGTG AAAGTTCCTG GGACCTGTTC GCGTGAGCTG AGCGGGGACG TGTATGCGAC GGCCAAAATT GGCTCTGAGA GGCACTTCGG CTGCTGCACT TGCCGTGAGC TCAGTGCTGC CTGGCCTCGC GCGGCCTCGG AATGTGGGGA GATGGGATTG GTCTGGAAGA GCCTGCCGGC CGGTGTCTGG GCGTGAGCAA TGGAGCAGAA TGCGCAGACA CCGCACCGGG GAGATTGGAA AGGAGAGGTA TTGCCGAGAG TGAACCTGGT CAACTTGTCG ACGAAAGCTG CGATCTGGAG CGATCTCCTG GGAGGCAGAT GAGACTGGCC CCCCCGAAGC GGAGAAAGCC GGCTGACCTG AGCAGCGATC GAGCGTGTGC CGCACACGCA CAACGCCTGG CTGCGGGTGG CGCCAGCTGA CTGAGTGAGC ACGGTGGCTG GTGGAAGGGA Nucleotide sequence of C. griseus Fadd cDNA. Underlined sequence denotes the open reading frame of FADD. MDPFLVLLHS VSGNLSSSDL LELKFLCRER VSKRKLERVQ SGLDLFSVLL EQNDLERTRT Death Effector Domain 61 GLLRELLASL RRHDLLQRLD DFEAGTAASA APGEADLRVA FDIVCDNVGR DWKRLARQLK 121 VSEAKIDGIE ERYPRSLSEQ VREALRVWKI AEREKATVAG LVKALRACRL NLVADLVEGR Death Domain Amino acid sequence of C. griseus FADD protein. The death effector domain (DED) is highlighted in light grey  while the death domain (DD) is highlighted in dark grey . A potential N-glycosylation site is located at Asn14. CHO MDPFLVLLHSVSGNLSSSDLLELKFLCRERVSKRKLERVQSGLDLFSVLLEQNDLERTRT Mouse Human 1 MDPFLVLLHSLSGSLSGNDLMELKFLCRERVSKRKLERVQSGLDLFTVLLEQNDLERGHT MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHT CHO Mouse Human 61 61 GLLRELLASLRRHDLLQRLDDFEAGTAASAAPGEADLRVAFDIVCDNVGRDWKRLARQLK GLLRELLASLRRHDLLQRLDDFEAGTATAAPPGEADLQVAFDIVCDNVGRDWKRLARELK ELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQLK CHO 121 Mouse 121 Human 121 VSEAKIDGIEERYPRSLSEQVREALRVWKIAEREKATVAGLVKALRACRLNLVADLVEGR VSEAKMDGIEEKYPRSLSERVRESLKVWKNAEKKNASVAGLVKALRTCRLNLVADLVEEA VSDTKIDSIEDRYPRNLTERVRESLRIWKNTEKENATVAHLVGALRSCQMNLVADLVQEV CHO 181 Mouse 181 Human 181 ------------------------QESVSKSENMSPVLRDSTVSSSETP QQARDLQNRSGAMSPMSWNSDASTSEAS Alignment of amino acid sequences of C. griseus, M. musculus (NM_010175.2) and H. sapien (NM_003824) FADD protein. Conserved residues are shaded in grey . 176 APPENDIX Fas Apoptosis Inhibitory Molecule (Faim) 61 121 181 241 301 361 421 481 541 601 661 721 781 841 GCCGCGAGAG CCTCTTTATA GATGGAGTCC GTGGATGGGA TTCTGTGTTG GCTTATGAGT TCAAAGACCA GAAAAAGATA GTAGATGATG GTCAGCAGCG ATCCCAGAGC GGACTTTCTA TCTGCAATGT GTGGAATCAT TAAAATATGG CTGCTGACTA ACCTGGAAAA ACAAGATTGA AGGAAGAGAT GAGCTGCGAA ATACCCTGGA CCAACACCTG CTATGGATGT GAACTGAAAC GGAAGAGAAG TCCCTCAGTG ATGGCTGTGG TTTTATTTTT AAAAATTTAT ACCAAACCCA CGTCGTGGGA AATGACAGAT ATTTGAGCAT AAGAAAAGAA AACCAAAGCC AATCGATGGG GGTACTGCAC ATGGTGCAAT ACACTTCAGT AGAAGGGATT ACTGCTGGTT TAATTAAATG TGTTACTGGA TTTTTAACTA TTCATATCTC TCAGGAGCCG CTTGTAGCTG GGGACCACAT TGGATGTTCA ACCATAAATA AAAAGCCTCA TTGGATGGCC GGTCAAAAAA GTTGGGAACC ATCCACACAC AGTGGGTTCT TGTTCACTGT AACTGTAATA CTGTAAAGTG ACCACAGTAA GTGGCGGAGC TTTGGGACGT CAGGCAAACG AATTGGTGGG TAGATGCTGT AGAAGTACAT AGGACTTAAG TGGAGACAGC ATGACTGTTA TCATTGTGGA GAGCTGAAGA GTACATATTG TTCCAATGGT TTTCTAATTC C GCCGGGCAGC TGCATTAAGT AGTTGTGTAC CAAAGAAACC CAGTGGTTTT GGAGAACAGA AGTTGTTTTG AGGCGAGTTT CATAAAAGCT TAACAGGGAG GGAGACATCA GTAGATTTAG CAAGAAAAAT AAATAGGAAA Nucleotide sequence of C. griseus Faim cDNA. Underlined sequence denotes the open reading frame of FAIM. MTDLVAVWDV ALSDGVHKIE FEHGTTSGKR VVSVDGKEEI RKEWMFKLV GKETFCVGAAK 61 PKATINIDAV SGFAYEYTLE IDGKSLKKYM KNRSKTTNTW VLHLDGQDL RVVLEKDTMDV WCNGQKMETA GEFVDDGTET HFSVGNHDCC IKAVSSGKRR EGIIHTLIV DNREIPELPQ Amino acid sequence of C. griseus FAIM protein. A potential N-glycosylation site is located at Asn92. CHO Mouse Human 1 MTDLVAVWDVALSDGVHKIEFEHGTTSGKRVVSVDGKEEIRKEWMFKLVGKETFCVGAAK MTDLVAVWDVALSDGVHKIEFEHGTTSGKRVVYVDGKEEIRREWMFKLVGKETFFVGAAK MTDLVAVWDVALSDGVHKIEFEHGTTSGKRVVYVDGKEEIRKEWMFKLVGKETFYVGAAK CHO Mouse Human 61 61 61 PKATINIDAVSGFAYEYTLEIDGKSLKKYMKNRSKTTNTWVLHLDGQDLRVVLEKDTMDV TKATINIDAISGFAYEYTLEIDGKSLKKYMENRSKTTSTWVLRLDGEDLRVVLEKDTMDV TKATINIDAISGFAYEYTLEINGKSLKKYMEDRSKTTNTWVLHMDGENFRIVLEKDAMDV CHO 121 Mouse 121 Human 121 WCNGQKMETAGEFVDDGTETHFSVGNHDCCIKAVSSGKRREGIIHTLIVDNREIPELPQ WCNGQKMETAGEFVDDGTETHFSVGNHGCYIKAVSSRKRKEGIIHTLIVDNREIPELTQ WCNGKKLETAGEFVDDGTETHFSIGNHDCYIKAVSSGKRKEGIIHTLIVDNREIPEIAS Alignment of amino acid sequences of C. griseus, M. musculus (NM_011810) and H. sapien (NM_018147.1) FAIM protein. Conserved residues are shaded in grey . 177 APPENDIX Apoptosis Linked Gene (Alg-2) 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 GCCCATGGCT AGCTGCGCTG GAGTGGAGTG GTTTAATCCA CGTGAACTTC CCGAACCTAT CTCAGGTTTT CAGACAAGGA GAGGTTGACA TTATGAGCAG GCACAGCATG AAGTACAGTT GTACATATGT ATACAAACAT GATTCTGCTC ACTTTTATTA TGCCTTACTT AGTAATGATC GCCTACTCCT CCAGACCAGA ATTTCAGACA GTGACTGTTA AGTGAATTTA GACCGGGACA GGCTACCGGC CGAGGGCAGA GACATATTCA TATCTCTCCA CAAAAAAGAC AGATGCTGTT GAATAAGCTG TGGGTTTGCT TCAAACTATC GCCAATAGGA TTTTAAAAAA CAGCCCAAAA ACCGCCCAGG GCTTCCTGTG ATGAGCTTCA GGTCAATCAT CAGGCGTGTG ACTCTGGGAT TCTCTGACCA TCGCATTTGA GACGCTATGA TGGTCTTCAG TGAAAATGCC CTTCCTGTAG GTTAGTGATT GACTAATTGT ATGTTCTTTT TTTTAAAATA GAGTTTATGT AAAAAAAAAA CCCGGGCGCC GAACGTCTTC GCAAGCATTA TTCTATGTTT GAAGTACATC GATTGACAAG GTTCCATGAC TGACTTCATC CACGGATCAG CATCGTATAA AAATCCCTTC GCTGTATAAT CTGTAGTGGC GCCACGAGGG CTAGCTGTCT ATATGGAACT ATTCATTGGA AAAAAAAAAA GGCCCCGGCC CAGCGGGTTG TCCAATGGTA GACCGAGAAA ACAGACTGGC AACGAGCTCA ATCCTCATCC CAGGGCTGCA GACGGCTGGA CCAGGCCCTG CCTGTGATGA TAATACTTGG ACCCTAGCTA GAAACCGAAT CTAATTCTGT TGCACAGAAG ATATGTAACA AAAAAAAA CTTCTGCTGG ATAAAGACAG CTTGGACTCC ACAAGGCTGG AGAATGTCTT AGCAAGCACT GCAAATTTGA TCGTCTTGCA TTCAGGTGTC TGAACAGCAA AACAGGGCAC GGACCTGGCT CACTGTTATA ATTGGTTCAG AGTTGAAAAT GCTTTTCATG TAAGCAATAA Nucleotide sequence of C. griseus Alg-2 cDNA. Underlined sequence denotes the open reading frame of ALG-2. MAAYSYRPGP GAGPGPSAGA ALPDQSFLWN VFQRVDKDRS GVISDNELQQ ALSNGTWTPF N-terminal hydrophobic domain EF domain 61 NPVTVRSIIS MFDRENKAGV NFSEFTGVWK YITDWQNVFR TYDRDNSGMI DKNELKQALS EF domain EF domain 121 GFGYRLSDQF HDILIRKFDR QGRGQIAFDD FIQGCIVLQR LTDIFRRYDT DQDGWIQVSY EF domain EF domain 181 EQYLSMVFSI V Amino acid sequence of C. griseus ALG-2 protein. The N-terminal hydrophobic domain is highlighted in dark grey  while the EF domains are highlighted in light grey . A potential N-glycosylation site is located at Asn14. CHO Human Mouse 1 MAAYSYRPGPGAGPGPSAGAALPDQSFLWNVFQRVDKDRSGVISDNELQQALSNGTWTPF MAAYSYRPGPGAGPGPAAGAALPDQSFLWNVFQRVDKDRSGVISDTELQQALSNGTWTPF MAAYSYRPGPGGGPGPAAGAALPDQSFLWNVFQRVDKDRSGVISDNELQQALSNGTWTPF CHO Human Mouse 61 61 61 NPVTVRSIISMFDRENKAGVNFSEFTGVWKYITDWQNVFRTYDRDNSGMIDKNELKQALS NPVTVRSIISMFDRENKAGVNFSEFTGVWKYITDWQNVFRTYDRDNSGMIDKNELKQALS NPVTVRSIISMFDRENKAGVNFSEFTGVWKYITDWQNVFRTYDRDNSGMIDKNELKQALS Continue on next page 178 CHO Human Mouse 121 121 121 GFGYRLSDQFHDILIRKFDRQGRGQIAFDDFIQGCIVLQRLTDIFRRYDTDQDGWIQVSY GFGYRLSDQFHDILIRKFDRQGRGQIAFDDFIQGCIVLQRLTDIFRRYDTDQDGWIQVSY GFGYRLSDQFHDILIRKFDRQGRGQIAFDDFIQGCIVLQRLTDIFRRYDTDQDGWIQVSY CHO Human Mouse 181 181 181 EQYLSMVFSIV EQYLSMVFSIV EQYLSMVFSIV Alignment of amino acid sequences of C. griseus, M. musculus (NM_011051.1) and H. sapien (NM_013232.2) ALG-2 protein. Conserved residues are shaded in grey . 179 APPENDIX Requiem 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 GGTATCAACG AGAATGTAGT ATTACAATGC CTGGAGTAGC TGGCCTCTGG ACCCACCTGA TGAAGAAAGA ACCCTCTGGA TTCCTGTCAC ATCTTGATGA AGAGTAAGGG ATAAGCCCTA ACCACTATGC CACCTACTCC TAGCCCTGCC CAGGGCAGCC GCCTGCAGTT AGTGCAAGTG ATGACTGTGA AAGGAAGTTG AGAACCAGAG CCTCTACCTT AACTGCCTTT CCTCAGGGAG GCTCTCCAAG GTTCAAATGG CCCTCCTCCC CTTGGAGCAC TGTTCTGCTT GTCCTGTGCT TCCCACAAAG GTGGTCTCTG CTTGTTCCCT GGGGACCACA CAAAATCCCC CAGGAGCCTC CTTGCACACT TGGCACTGAC TCCTTGGCTG CTTAGTTCCT AAAAAAAAAA AAAAAAAAAA CAGCCTCCCG GAAGCTCCTT CCGCCTCTGT CCAGAGCAAT ACAGTTGTAC GGATCCCAGG GGGGCTTATA GAAACGAGGT CAACAGTCGA TGAAGACTAT TGTGAGCAGT TGCCTGTGAC CTATTCCCAC TGTTTCCCAG CAACAACTAC TGAGGAGCTA CACCCCCGTG CTGCAACCTC CCGTGGCTAC GAGCTGCCAC CTCCTCCTGA GGTTTTCATA GGCAACAGCA TAAGGAGCAG AAGTGCATTC GTTCCTCTAA CCTCTGGTGT CTGGAGTGAT CACTTTGCCT TGCACTGTGA AGACAAGCAG GCAATCCAAA TTGGTTTTGT AGAAGTTGGA TTTTCGTTCT TGCCTCCTAC AGCTAGCAGC ATTAAGTAGG TGGGCAGGCC GTATCCAAAA AAAAAAAAAA AAAAAAAAAA GCGGGAGGGA GGGGAACAGT GCTGAGCGTA TGTTATATCT TCCTACCCTG CTTTCCTTTC TCTCAGGATG GCTCCAGATC GCACGGAAGC GAAGAAGATA GCCCGGAAGA ATTTGTGGAA CTGGCTGAGG AGGTCTGAGG TGTGACTTCT GTGTCCTGTT ATGATGGCGG TGCGGCACTT CACATGTACT CTGTGTCTGG TGTGCCACCC CCCCTCTTCT CAGGGAAGGT CATGCTGCCC ACTCTGCTTG GAAGTATGAG CCAGAGTTTT CAGACTGAGG CTGCCAAAGC TGCTCTCAGG CTTCACCTGC CATTTCCCAT GGGGGAGAGG TAATGTGCCT TCCTGGACCT TGTGTATCCT TGGTAAAGTC AGGGGACAGT CTGACTTGTT CATTAGTAAG AAAAAAAAAA AA GAGGAGCAGG ACTACAAAGA GCGTGCGTCT GGATGGAAAA CCCGGCGCTG CATCTATTAA GCAGCAGTTT CCCGTGTTGA GGATCCTTGA CTCCAAAACG AACTGGATGC AACGCTACAA AGGAGGGAGA AGCAGAAATC GCCTGGGAGA CTGACTGTGG CTGTGAAGAC CGGAGAATGA GTCTCACCTC ATCTGCTGAA GGCTCCCCAC TCTTCTTCTT GGCAACTCTT CAGGCTGATC CCTTGGGCCC AGCAGCTCAC ACCCCAGGGG TGGCACTTGC AGTCCTGTGT CACCTCCTGG CCTTCCCGTG CCTCAGACTT GACAATGTCA GTTTTTTAAC GGGCATTCAG GGCTCCCAGG TTCTTTCCCT CCATGCCAGA TTCTGCAAAG AATAAACATT AAAAAAAAAA GAAGATGGCG TGCCATGGAA GCCTTTCTTG GCGACACCGG GCGTAAAAAG ACCAGACACA AGAGGCTCTA TGATGACAGC ACCTGATGAC TCGGGGAAAG TTCCATCCTG GAATCGACCT GGACAAAGAA CAAGAAAGGA CTCAAAAATC CCGCTCAGGG CTACCGCTGG CGACCAGCTG ATCCATGTCG GGAGAAAGCG ACACCTAAGG TCACTCTGGT GACTGCCTCT TGTGGGCCCA AAGTCCCTGG TTGTCTCAAG TGAGCCAGGC TAGGACCCTT CTTCTGTCAT CTCTGTCCTG CTTGGCTGGC TTGAGTCTTC GGGGGCCCTG TCGATAAAAA CCTCCTGCCC AGAGAGGATG GATTTTTGTT ACACTCTGGA TTGAGGCCCC TTTACACAGA AAAAAAAAAA GCTGTGGTGG CAGTGCCACA GACTCACAGA GGACCAGGAT CGTCGAGCTC GACCAGACCC CTGCGTACTG CTGGGCGAGT TTCCTAGATG GGGAAATCCA GAGGACCGTG TGCCTCAGTT GACTCTCAAC CCTGATGGAT AACAAGAAGA CACCCGTCCT CAGTGCATCG CTCTTCTGTG GAGCCTCCTG TCCATCTACC CTGTTGCTCT AGTTCTGCCC GGTCCCAAGC GCTTCTCTCT TAATCACAGG CCTGGCCTAC CTAACCTTTG TCCTACCCCT GCTACATGGG TTCTGCCCAG GCGCTCACAG TGCCTCCTTC CCAGAAGCTT TGCCTACCTC TTAACTGAAT GTCCCCTTTC TCCTGCTTAG ATGGCCTTCC TCCTCCTATC AAAAAAAAAA AAAAAAAAAA Nucleotide sequence of C. griseus Requiem cDNA. Underlined sequence denotes the open reading frame of REQUIEM. 180 MAAVVENVVK LLGEQYYKDA MEQCHNYNAR LCAERSVRLP FLDSQTGVAQ SNCYIWMEKR 61 HRGPGLASGQ LYSYPARRWR KKRRAHPPED PRLSFPSIKP DTDQTLKKEG LISQDGSSLE 121 ALLRTDPLEK RGAPDPRVDD DSLGEFPVTN SRARKRILEP DDFLDDLDDE DYEEDTPKRR 181 GKGKSKSKGV SSARKKLDAS ILEDRDKPYA CDICGKRYKN RPCLSYHYAY SHLAEEEGED Zinc finger C2H2-type domain 241 KEDSQPPTPV SQRSEEQKSK KGPDGLALPN NYCDFCLGDS KINKKTGQPE ELVSCSDCGR Zinc finger PHD-type domain 301 SGHPSCLQFT PVMMAAVKTY RWQCIECKCC NLCGTSENDD QLLFCDDCDR GYHMYCLTSS Zinc finger PHD-type domain 361 MSEPPEGSWS CHLCLDLLKE KASIYQNQSS S Amino acid sequence of C. griseus REQUIEM protein. The zinc finger C2H2-type domain is highlighted in dark grey  while the zinc finger PHD-type domains are highlighted in light grey . There is a potential N-glycosylation site at Asn387. CHO Mouse Human 1 MAAVVENVVKLLGEQYYKDAMEQCHNYNARLCAERSVRLPFLDSQTGVAQSNCYIWMEKR MAAVVENVVKLLGEQYYKDAMEQCHNYNARLCAERSVRLPFLDSQTGVAQSNCYIWMEKR MAAVVENVVKLLGEQYYKDAMEQCHNYNARLCAERSVRLPFLDSQTGVAQSNCYIWMEKR CHO Mouse Human 61 61 61 HRGPGLASGQLYSYPARRWRKKRRAHPPEDPRLSFPSIKPDTDQTLKKEGLISQDGSSLE HRGPGLASGQLYSYPARRWRKKRRAHPPEDPRLSFPSIKPDTDQTLKKEGLISQDGSSLE HRGPGLASGQLYSYPARRWRKKRRAHPPEDPRLSFPSIKPDTDQTLKKEGLISQDGSSLE CHO 121 Mouse 121 Human 121 ALLRTDPLEKRGAPDPRVDDDSLGEFPVTNSRARKRILEPDDFLDDLDDEDYEEDTPKRR ALLRTDPLEKRGAPDPRVDDDSLGEFPVSNSRARKRIIEPDDFLDDLDDEDYEEDTPKRR ALLRTDPLEKRGAPDPRVDDDSLGEFPVTNSRARKRILEPDDFLDDLDDEDYEEDTPKRR CHO 181 Mouse 181 Human 181 GKGKSKSKGVSSARKKLDASILEDRDKPYACDICGKRYKNRPCLSYHYAYSHLAEEEGED GKGKSKSKGVSSARKKLDASILEDRDKPYACDICGKRYKNRPGLSYHYAHSHLAEEEGED GKGKSKGKGVGSARKKLDASILEDRDKPYACDICGKRYKNRPGLSYHYAHSHLAEEEGED CHO 241 Mouse 241 Human 241 KEDSQPPTPVSQRSEEQKSKKGPDGLALPNNYCDFCLGDSKINKKTGQPEELVSCSDCGR KEDSRPPTPVSQRSEEQKSKKGPDGLALPNNYCDFCLGDSKINKKTGQPEELVSCSDCGR KEDSQPPTPVSQRSEEQKSKKGPDGLALPNNYCDFCLGDSKINKKTGQPEELVSCSDCGR CHO 301 Mouse 301 Human 301 SGHPSCLQFTPVMMAAVKTYRWQCIECKCCNLCGTSENDDQLLFCDDCDRGYHMYCLTSS SGHPSCLQFTPVMMAAVKTYRWQCIECKCCNLCGTSENDDQLLFCDDCDRGYHMYCLTPS SGHPSCLQFTPVMMAAVKTYRWQCIECKCCNICGTSENDDQLLFCDDCDRGYHMYCLTPS CHO 361 Mouse 361 Human 361 MSEPPEGSWSCHLCLDLLKEKASIYQNQSSS MSEPPEGSWSCHLCLDLLKEKASIYQNQNSS MSEPPEGSWSCHLCLDLLKEKASIYQNQNSS Alignment of amino acid sequences of C. griseus, M. musculus (NM_011262.1) and H. sapien (NM_006268.3) REQUIEM protein. Conserved residues are shaded in grey . 181 APPENDIX Publications: Wong CFD, Wong TKK, Goh LT, Heng CK and Yap MGS. 2005. Impact of Dynamic Online Fed-Batch 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 and Yap MGS. 2006. Transcriptional Profiling of Apoptotic Pathways in Batch and Fed-batch CHO Cell Cultures. Biotechnol Bioeng. 94: 373-382 Wong CFD, Wong TKK, Heng CK and Yap MGS. 2006. Targeting Early Apoptotic Genes in Batch and Fed-Batch CHO cell Cultures. Biotechnol Bioeng. 95: 350-361 182 [...]... sialic acid 10 6 content DISCUSSIONS 10 7 4.4 .1 Improving FBC through use of dynamic online feeding 10 7 4.4 .1 N-Glycosylation in FBC 10 8 10 9 CONCLUSION CHAPTER 5 Transcriptional Profiling of Apoptotic Pathways in Batch and Fed- batch CHO Cell Cultures INTRODUCTION 11 1 11 1 11 RESULTS 11 1 5 .1 111 Apoptosis Signaling During BC and FBC (A) Up-regulation of FasL and Fadd in the Death Receptor- 11 5 mediated... cell BC and FBC 12 0 5.2.2 Strategies to delay onset of apoptosis in culture 12 2 CONCLUSIONS CHAPTER 6 12 4 Targeting Early Apoptotic Genes in Batch and Fed- 12 6 Batch CHO Cell Cultures INTRODUCTION 12 6 RESULTS 12 7 6 .1 Cloning of Apoptotic Homologs from CHO cells 12 7 6.2 Creation of Gene Targeted CHO (CHO GT) cell lines 12 9 6.3 CHO GT Cells in BC 13 1 6.3 .1 Growth of CHO GT Transfected Pools in BC 13 1 6.3.2... pool in BC 13 2 Figure 6.5 CHO GTKD cells in BC The viable cell density, viability and percentage of apoptotic cells of CHO GTKD ALG-2 pool and CHO GTKD REQUIEM pool in BC 13 3 Figure 6.6 Recombinant human IFN-γ yields of CHO GT cells in BC 13 4 Figure 6.7 Specific productivities of recombinant human IFN-γ in CHO GT cells in BC 13 5 Figure 6.8 Caspase-8, -9 and –3 activities during BC of CHO GTO cell lines... cell lines 12 9 96 18 1 INTRODUCTION 1. 1 Background One of the challenges faced by large-scale production of therapeutic proteins is the need to achieve high cell density while maintaining high culture viability in order to obtain high recombinant glycoprotein yield and quality In most of these proceses, batch (BC) and fed- batch (FBC) cultures are the main culture modes used for recombinant protein production... Activities of Caspases in BC 13 5 12 6.4 CHO GT Cell Lines in FBC 13 8 6.5 N-glycosylation Quality of IFN-γ Produced by CHO GT cells during FBC 14 0 6.5 .1 Macro-heterogeneity of recombinant IFN-γ 14 0 6.5 .1 Micro-heterogeneity of recombinant IFN-γ 14 1 6.5 .1 Sialylation of recombinant IFN-γ 14 3 DISCUSSION 14 4 6.6 .1 Targeting of Fadd, Faim, Alg-2 and Requiem to Prolong BC and FBC 14 4 6.6 .1 Strategies to Enhance... GTO cell lines 13 5 Figure 6.9 Caspase-8, -9 and –3 activities during BC of CHO GTKD cell lines 13 7 Figure 6 .10 Viable cell densities of CHO GT cell lines in FBC 13 9 Figure 6 .11 Enhanced recombinant human IFN-γ yields in CHO GT cell lines during FBC 14 0 Figure 6 .12 Site-occupancy of IFN-γ purified from FBC of CHO GT cells 14 1 Figure 6 .13 Micro-heterogeneity of complex-type glycans of recombinant human... harvested from CHO GT cells 14 2 Figure 6 .14 Sialylation of recombinant IFN-γ in CHO GT cell lines during mid-exponential, stationary and death phase of FBC 14 3 17 LIST OF TABLES: Page Table 2 .1 Examples of recombinant therapeutics proteins produced in CHO cells 24 Table 2.2 Effects of the anti-apoptosis genetic engineering on CHO cell lines 41 Table 3 .1 List of N-linked oligosaccharide standards and their... Signaling during BC and FBC (A) Up-regulation of Bim and Bad in the Mitochondrial-mediated 11 7 Apoptosis Signaling during BC and FBC (A) Down-regulation of Ire -1 and up-regulation of Alg-2 in the 11 8 Endoplasmic Reticulum (ER)-mediated Apoptosis Signaling during BC and FBC (D) Differential expression of inhibitors of apoptosis proteins 11 9 DISCUSSIONS 12 0 5.2 .1 Apoptosis-related Cell Death in CHO cell... 10 6 Figure 5 .1 BC and FBC for expression profiling using microarrays 11 2 Figure 5.2 Apoptosis-related genes regulated during BC and FBC of CHO cells 11 3 Figure 5.3 Validation of microarray expression profiles of apoptosis signaling genes across exponential, stationary and death phases of BC and FBC 11 4 Figure 5.4 Apoptosis signaling in BC and FBC of CHO cells 11 6 Figure 6 .1 Apoptosis signaling via death... of the thesis involved: (1) Developing an enhanced high- density FBC based on a dynamic online feeding strategy for CHO cells producing recombinant human interferon gamma (IFN-γ) and determining the impact on IFN-γ production and glycosylation quality, (2) Examining the signaling pathways that are responsible for apoptosis induction in BC and FBC processes using transcriptome analysis and (3) Based . CHO (CHO GT) cell lines 6.3 CHO GT Cells in BC 6.3 .1 Growth of CHO GT Transfected Pools in BC 6.3.2 Proteolytic Activities of Caspases in BC 11 1 11 1 11 5 11 7 11 8 11 9 12 0 12 0 12 2 12 4 12 6 12 6 12 7 12 7 12 9 13 1 13 1 13 5 13 6.4. Augmenting recombinant production using epigenetic gene silencing Abbreviations References 13 8 14 0 14 0 14 1 14 3 14 4 14 4 14 5 14 6 14 8 14 8 15 0 15 0 15 1 15 1 15 2 15 3 15 3 15 5 15 7 14 APPENDICES Appendix 1. Medicine National University of Singapore 2006 2 Enhancing Recombinant Glycoprotein Yield & Quality Using Novel CHO GT Cells in High Density Fed- batch Cultures SUMMARY Chinese Hamster Ovary (CHO)