Skip to main content
SpringerLink
Log in
Book cover

Heterologous Protein Production in CHO Cells pp 1–23Cite as

Strategies and Considerations for Improving Expression of “Difficult to Express” Proteins in CHO Cells

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1603))

Abstract

Despite substantial advances in the field of mammalian expression, there are still proteins that are characterized as difficult to express. Determining the expression bottleneck requires troubleshooting techniques specific for the given molecule and host. The complex array of intracellular processes involved in protein expression includes transcription, protein folding, post-translation processing, and secretion. Challenges in any of these steps could result in low protein expression, while the inherent properties of the molecule itself may limit its production via mechanisms such as cytotoxicity or inherent instability. Strategies to identify the rate-limiting step and subsequently improve expression and production are discussed here.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   179.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Ohya T, Hayashi T, Kiyama E et al (2008) Improved production of recombinant human antithrombin III in Chinese hamster ovary cells by ATF4 overexpression. Biotechnol Bioeng 100:317–324. doi:10.1002/bit.21758

    Article  CAS  PubMed  Google Scholar 

  2. Novo JB, Morganti L, Moro AM et al (2012) Generation of a Chinese hamster ovary cell line producing recombinant human glucocerebrosidase. J Biomed Biotechnol 2012:875383. doi:10.1155/2012/875383

    Article  PubMed  PubMed Central  Google Scholar 

  3. Le Fourn V, Girod P-A, Buceta M et al (2014) CHO cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion. Metab Eng 21:91–102. doi:10.1016/j.ymben.2012.12.003

    Article  CAS  PubMed  Google Scholar 

  4. Jiang Z, Huang Y, Sharfstein ST (2006) Regulation of recombinant monoclonal antibody production in chinese hamster ovary cells: a comparative study of gene copy number, mRNA level, and protein expression. Biotechnol Prog 22:313–318

    Article  CAS  PubMed  Google Scholar 

  5. Nishimiya D, Mano T, Miyadai K et al (2013) Overexpression of CHOP alone and in combination with chaperones is effective in improving antibody production in mammalian cells. Appl Microbiol Biotechnol 97:2531–2539. doi:10.1007/s00253-012-4365-9

    Article  CAS  PubMed  Google Scholar 

  6. Mead EJ, Chiverton LM, Smales CM, von der Haar T (2009) Identification of the limitations on recombinant gene expression in CHO cell lines with varying luciferase production rates. Biotechnol Bioeng 102:1593–1602. doi:10.1002/bit.22201

    Article  CAS  PubMed  Google Scholar 

  7. Hu Z, Guo D, Yip SSM et al (2013) Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11-derived dihydrofolate reductase deficient host cell. Biotechnol Prog 29:980–985. doi:10.1002/btpr.1730

    Article  CAS  PubMed  Google Scholar 

  8. Alves C, Gilbert A, Dalvi S et al (2015) Integration of cell line and process development to overcome the challenge of a difficult to express protein. Biotechnol Prog:1–11. doi:10.1002/btpr.2091

  9. Lattenmayer C, Trummer E, Schriebl K et al (2007) Characterisation of recombinant CHO cell lines by investigation of protein productivities and genetic parameters. J Biotechnol 128:716–725. doi:10.1016/j.jbiotec.2006.12.016

    Article  CAS  PubMed  Google Scholar 

  10. Reisinger H, Steinfellner W, Stern B et al (2008) The absence of effect of gene copy number and mRNA level on the amount of mAb secretion from mammalian cells. Appl Microbiol Biotechnol 81:701–710. doi:10.1007/s00253-008-1701-1

    Article  CAS  PubMed  Google Scholar 

  11. Chusainow J, Yang YS, Yeo JHM et al (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 102:1182–1196. doi:10.1002/bit.22158

    Article  CAS  PubMed  Google Scholar 

  12. Hindson BJ, Ness KD, Masquelier D a., et al. (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83:8604–8610. doi: 10.1021/ac202028g

  13. Huang Y, Li Y, Wang YG et al (2007) An efficient and targeted gene integration system for high-level antibody expression. J Immunol Methods 322:28–39. doi:10.1016/j.jim.2007.01.022

    Article  CAS  PubMed  Google Scholar 

  14. Wright C, Estes SD (2014) Pharmaceutical next-generation bioprocess: an industry perspective of how the ‘ omics era will affect future biotherapeutic development. Pharm Bioprocess 2:371–375

    Article  Google Scholar 

  15. Büssow K (2015) Stable mammalian producer cell lines for structural biology. Curr Opin Struct Biol 32:81–90. doi:10.1016/j.sbi.2015.03.002

    Article  PubMed  Google Scholar 

  16. Turan S, Galla M, Ernst E et al (2011) Recombinase-mediated cassette exchange (RMCE): traditional concepts and current challenges. J Mol Biol 407:193–221. doi:10.1016/j.jmb.2011.01.004

    Article  CAS  PubMed  Google Scholar 

  17. Baser B, Spehr J, Büssow K, van den Heuvel J (2015) A method for specifically targeting two independent genomic integration sites for co-expression of genes in CHO cells. Methods 95:3–12. doi:10.1016/j.ymeth.2015.11.022

    Article  PubMed  Google Scholar 

  18. Turan S, Zehe C, Kuehle J et al (2013) Recombinase-mediated cassette exchange (RMCE) - a rapidly-expanding toolbox for targeted genomic modifications. Gene 515:1–27. doi:10.1016/j.gene.2012.11.016

    Article  CAS  PubMed  Google Scholar 

  19. Inao T, Kawabe Y, Yamashiro T et al (2015) Improved transgene integration into the Chinese hamster ovary cell genome using the Cre-loxP system. J Biosci Bioeng 120:99–106. doi:10.1016/j.jbiosc.2014.11.019

    Article  CAS  PubMed  Google Scholar 

  20. Kawabe Y, Shimomura T, Huang S et al (2016) Targeted transgene insertion into the CHO cell genome using Cre recombinase-incorporating integrase-defective retroviral vectors. Biotechnol Bioeng:1600–1610. doi:10.1002/bit.25923

  21. Sakuma T, Takenaga M, Kawabe Y et al (2015) Homologous recombination-independent large gene cassette knock-in in CHO cells using TALEN and MMEJ-directed donor plasmids. Int J Mol Sci 16:23849–23866. doi:10.3390/ijms161023849

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bachu R, Bergareche I, Chasin L a. (2015) CRISPR-Cas targeted plasmid integration into mammalian cells via non-homologous end joining. Biotechnol Bioeng 112:2154–2162. doi: 10.1002/bit.25629

  23. Lee JS, Kallehauge TB, Pedersen LE, Kildegaard HF (2015) Site-specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway. Sci Rep 5:8572. doi:10.1038/srep08572

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Peterson CL, Laniel M (2004) Histones and histone modifications. Curr Biol 14:546–551

    Article  Google Scholar 

  25. Reinhart D, Sommeregger W, Debreczeny M et al (2013) Characterization of recombinant IgA producing CHO cell lines by qPCR. BMC Proc 7:P114. doi:10.1186/1753-6561-7-S6-P114

    Article  PubMed Central  Google Scholar 

  26. Schlatter S, Stansfield SH, Dinnis DM et al (2005) On the Optimal Ratio of Heavy to Light Chain Genes for Efficient Recombinant Antibody Production by CHO Cells. Biotechnol Prog 21:122–133

    Article  CAS  PubMed  Google Scholar 

  27. Ho SCL, Koh EYC, van Beers M et al (2013) Control of IgG LC:HC ratio in stably transfected CHO cells and study of the impact on expression, aggregation, glycosylation and conformational stability. J Biotechnol 165:157–166. doi:10.1016/j.jbiotec.2013.03.019

    Article  CAS  PubMed  Google Scholar 

  28. Osterlehner A, Simmeth S, Göpfert U (2011) Promoter methylation and transgene copy numbers predict unstable protein production in recombinant Chinese hamster ovary cell lines. Biotechnol Bioeng 108:2670–2681. doi:10.1002/bit.23216

    Article  CAS  PubMed  Google Scholar 

  29. Wippermann A, Rupp O, Brinkrolf K et al (2015) The DNA methylation landscape of Chinese hamster ovary (CHO) DP-12 cells. J Biotechnol 199:38–46. doi:10.1016/j.jbiotec.2015.02.014

    Article  CAS  PubMed  Google Scholar 

  30. De Carvalho DD, You JS, Jones PA (2011) DNA methylation and cellular reprogramming. Trends Cell Biol 20:609–617. doi:10.1016/j.tcb.2010.08.003.DNA

    Article  Google Scholar 

  31. Clark SJ, Harrison J, Paul CL et al (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22:2990–2997

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Adorján P, Distler J, Lipscher E et al (2002) Tumour class prediction and discovery by microarray-based DNA methylation analysis. Nucleic Acids Res 30:e21. doi:10.1016/S0959-8049(01)80570-0

    Article  PubMed  PubMed Central  Google Scholar 

  33. Backliwal G, Hildinger M, Kuettel I et al (2008) Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures. Biotechnol Bioeng 101:182–189. doi:10.1002/bit.21882

    Article  CAS  PubMed  Google Scholar 

  34. Kouzarides T (2000) Acetylation: a regulatory modification to rival phosphorylation? EMBO J 19:1176–1179. doi:10.1093/emboj/19.6.1176

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Jiang Z, Sharfstein ST (2008) Sodium butyrate stimulates monoclonal antibody over-expression in CHO cells by improving gene accessibility. Biotechnol Bioeng 100:189–194. doi:10.1002/bit.21726

    Article  CAS  PubMed  Google Scholar 

  36. Kyoung MIN (2007) Correlation between enhancing effect of sodium butyrate on specific productivity and mRNA transcription level in recombinant chinese hamster ovary cells producing antibody. J Microbiol Biotechnol 17:1036–1040

    Google Scholar 

  37. Yang WC, Lu J, Nguyen NB et al (2014) Addition of valproic acid to CHO cell fed-batch cultures improves monoclonal antibody titers. Mol Biotechnol 56:421–428. doi:10.1007/s12033-013-9725-x

    Article  CAS  PubMed  Google Scholar 

  38. Kim NS, Lee GM (2002) Inhibition of sodium butyrate-induced apoptosis in recombinant Chinese hamster ovary cells by constitutively expressing antisense RNA of caspase-3. Biotechnol Bioeng 78:217–228. doi:10.1002/bit.10191

    Article  CAS  PubMed  Google Scholar 

  39. Barnes LM, Dickson AJ (2006) Mammalian cell factories for efficient and stable protein expression. Curr Opin Biotechnol 17:381–386. doi:10.1016/j.copbio.2006.06.005

    Article  CAS  PubMed  Google Scholar 

  40. Kwaks THJ, Otte AP (2006) Employing epigenetics to augment the expression of therapeutic proteins in mammalian cells. Trends Biotechnol 24:137–142. doi:10.1016/j.tibtech.2006.01.007

    Article  CAS  PubMed  Google Scholar 

  41. Maksimenko O, Gasanov NB, Georgiev P, Lines TPC (2015) Regulatory elements in vectors for efficient generation of cell Lines producing target proteins. Acta Naturae 7:15–26

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Harraghy N, Gaussin A, Mermod N (2008) Sustained transgene expression using MAR elements. Curr Gene Ther 8:353–366. doi:10.2174/156652308786071032

    Article  CAS  PubMed  Google Scholar 

  43. Harraghy N, Buceta M, Regamey A et al (2012) Using matrix attachment regions to improve recombinant protein production. Methods Mol Biol 801:93–110. doi:10.1007/978-1-61779-352-3_7

    Article  CAS  PubMed  Google Scholar 

  44. Betts Z, Croxford AS, Dickson AJ (2015) Evaluating the interaction between UCOE and DHFR-linked amplification and stability of recombinant protein expression. Biotechnol Prog 31:1014–1025. doi:10.1002/btpr.2083

    Article  CAS  PubMed  Google Scholar 

  45. Kwaks THJ, Barnett P, Hemrika W et al (2003) Identification of anti-repressor elements that confer high and stable protein production in mammalian cells. Nat Biotechnol 21:553–558. doi:10.1038/nbt814

    Article  CAS  PubMed  Google Scholar 

  46. Otte AP, Kwaks THJ, Van Blokland RJM et al (2007) Various expression-augmenting DNA elements benefit from STAR-select, a novel high stringency selection system for protein expression. Biotechnol Prog 23:801–807. doi:10.1021/bp070107r

    Article  CAS  PubMed  Google Scholar 

  47. Mead EJJ, Smales CMM (2011) mRNA translation and recombinant gene expression from mammalian cell expression systems. Compr Biotechnol 1:403–409. doi:10.1016/B978-0-08-088504-9.00043-X

    Article  Google Scholar 

  48. Mead EJ, Masterton RJ, Feary M et al (2015) Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity. Biochem J 472:261–273. doi:10.1042/BJ20150928

    Article  CAS  PubMed  Google Scholar 

  49. Chong WPK, Sim LC, Wong KTK, Yap MGS (2009) Enhanced IFN c production in adenosine-treated CHO cells: a mechanistic study. Biotechnol Prog 25:866–873. doi:10.1021/bp.118

    Article  CAS  PubMed  Google Scholar 

  50. Dreesen IA, Fussenegger M (2011) Ectopic expression of human mTOR increases viability, robustness, cell size, proliferation, and antibody production of chinese hamster ovary cells. Biotechnol Bioeng 108:853–866. doi:10.1002/bit.22990

    Article  CAS  PubMed  Google Scholar 

  51. Gustafsson C, Govindarajan S, Minshull J (2004) Codon bias and heterologous protein expression. Trends Biotechnol 22:346–353. doi:10.1016/j.tibtech.2004.04.006

    Article  CAS  PubMed  Google Scholar 

  52. Welch M, Villalobos a., Gustafsson C, Minshull J (2009) You’re one in a googol: optimizing genes for protein expression. J R Soc Interface 6:S467–S476. doi: 10.1098/rsif.2008.0520.focus

  53. Chung BK-S, Yusufi FNK, Mariati, et al. (2013) Enhanced expression of codon optimized interferon gamma in CHO cells. J Biotechnol 167:326–333. doi: 10.1016/j.jbiotec.2013.07.011

  54. Kozak M (1991) Structural features in eukaryotic mRNAs that modulate the initiation of translation. J Biol Chem 266:19867–19870

    CAS  PubMed  Google Scholar 

  55. Nishimiya D (2014) Proteins improving recombinant antibody production in mammalian cells. Appl Microbiol Biotechnol 98:1031–1042. doi:10.1007/s00253-013-5427-3

    Article  CAS  PubMed  Google Scholar 

  56. Pybus LP, Dean G, West NR et al (2013) Model-directed engineering of “difficult-to-express” monoclonal antibody production by Chinese hamster ovary cells. Biotechnol Bioeng xxx:1–14. doi:10.1002/bit.25116

    Google Scholar 

  57. Johari YB, Estes SD, Alves CS et al (2015) Integrated cell and process engineering for improved transient production of a “difficult-to-express” fusion protein by CHO cells. Biotechnol Bioeng 112:2527–2542. doi:10.1002/bit.25687

    Article  CAS  PubMed  Google Scholar 

  58. Chollet ME, Skarpen E, Iversen N et al (2015) The chemical chaperone sodium 4-phenylbutyrate improves the secretion of the protein CA267T mutant in CHO-K1 cells trough the GRASP55 pathway. Cell Biosci 5:57. doi:10.1186/s13578-015-0048-4

    Article  PubMed  PubMed Central  Google Scholar 

  59. De Almeida SF, Picarote G, Fleming JV et al (2007) Chemical chaperones reduce endoplasmic reticulum stress and prevent mutant HFE aggregate formation. J Biol Chem 282:27905–27912. doi:10.1074/jbc.M702672200

    Article  CAS  PubMed  Google Scholar 

  60. Hwang S-J, Jeon C-J, Cho SM et al (2011) Effect of chemical chaperone addition on production and aggregation of recombinant flag-tagged COMP-angiopoietin 1 in Chinese hamster ovary cells. Biotechnol Prog 27:587–591. doi:10.1002/btpr.579

    Article  CAS  PubMed  Google Scholar 

  61. Liu C-H, Chen L-H (2007) Promotion of recombinant macrophage colony stimulating factor production by dimethyl sulfoxide addition in Chinese hamster ovary cells. J Biosci Bioeng 103:45–49. doi:10.1263/jbb.103.45

    Article  CAS  PubMed  Google Scholar 

  62. Liu C-H, Chen L-H (2007) Enhanced recombinant M-CSF production in CHO cells by glycerol addition: model and validation. Cytotechnology 54:89–96. doi:10.1007/s10616-007-9078-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Chakravarthi S, Jessop CE, Bulleid NJ (2006) The role of glutathione in disulphide bond formation and endoplasmic-reticulum-generated oxidative stress. EMBO Rep 7:271–275. doi:10.1038/sj.embor.7400645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Jing Y, Borys M, Nayak S et al (2012) Identification of cell culture conditions to control protein aggregation of IgG fusion proteins expressed in Chinese hamster ovary cells. Process Biochem 47:69–75. doi:10.1016/j.procbio.2011.10.009

    Article  CAS  Google Scholar 

  65. Rezaei M, Zarkesh-Esfahani SH, Gharagozloo M (2013) The effect of different media composition and temperatures on the production of recombinant human growth hormone by CHO cells. Res Pharm Sci 8:211–217

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Vergara M, Berrios J, Martínez I et al (2015) Endoplasmic reticulum-associated rht-PA processing in CHO cells: Influence of mild hypothermia and specific growth rates in batch and Chemostat cultures. PLoS One 10:e0144224. doi:10.1371/journal.pone.0144224

    Article  PubMed  PubMed Central  Google Scholar 

  67. Walsh G, Jefferis R (2006) Post-translational modifications in the context of therapeutic proteins. Nat Biotechnol 24:1241–1252. doi:10.1038/nbt1252

    Article  CAS  PubMed  Google Scholar 

  68. De Leon GM, Wlaschin KF, Nissom PM et al (2007) Comparative transcriptional analysis of mouse hybridoma and recombinant Chinese hamster ovary cells undergoing butyrate treatment. J Biosci Bioeng 103:82–91. doi:10.1263/jbb.103.82

    Article  Google Scholar 

  69. Sung YH, Song YJ, Lim SW et al (2004) Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells. J Biotechnol 112:323–335. doi:10.1016/j.jbiotec.2004.05.003

    Article  CAS  PubMed  Google Scholar 

  70. Kantardjieff A, Jacob NM, Yee JC et al (2010) Transcriptome and proteome analysis of Chinese hamster ovary cells under low temperature and butyrate treatment. J Biotechnol 145:143–159. doi:10.1016/j.jbiotec.2009.09.008

    Article  CAS  PubMed  Google Scholar 

  71. Knappskog S, Ravneberg H, Gjerdrum C et al (2007) The level of synthesis and secretion of Gaussia princeps luciferase in transfected CHO cells is heavily dependent on the choice of signal peptide. J Biotechnol 128:705–715. doi:10.1016/j.jbiotec.2006.11.026

    Article  CAS  PubMed  Google Scholar 

  72. Hegde RS, Bernstein HD (2006) The surprising complexity of signal sequences. Trends Biochem Sci 31:563–571. doi:10.1016/j.tibs.2006.08.004

    Article  CAS  PubMed  Google Scholar 

  73. Kober L, Zehe C, Bode J (2013) Optimized signal peptides for the development of high expressing CHO cell lines. Biotechnol Bioeng 110:1164–1173. doi:10.1002/bit.24776

    Article  CAS  PubMed  Google Scholar 

  74. Haryadi R, Ho S, Kok YJ et al (2015) Optimization of heavy chain and light chain signal peptides for high level expression of therapeutic antibodies in CHO cells. PLoS One 10:e0116878. doi:10.1371/journal.pone.0116878

    Article  PubMed  PubMed Central  Google Scholar 

  75. Petersen TN, Brunak S, von Heijne G, Nielsen H (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786. doi:10.1038/nmeth.1701

    Article  CAS  PubMed  Google Scholar 

  76. Frank K, Sippl MJ (2008) High-performance signal peptide prediction based on sequence alignment techniques. Bioinformatics 24:2172–2176. doi:10.1093/bioinformatics/btn422

    Article  CAS  PubMed  Google Scholar 

  77. Stoops J, Byrd S, Hasegawa H (2012) Russell body inducing threshold depends on the variable domain sequences of individual human IgG clones and the cellular protein homeostasis. Biochim Biophys Acta 1823:1643–1657. doi:10.1016/j.bbamcr.2012.06.015

    Article  CAS  PubMed  Google Scholar 

  78. Hasegawa H, Woods CE, Kinderman F et al (2014) Russell body phenotype is preferentially induced by IgG mAb clones with high intrinsic condensation propensity: relations between the biosynthetic events in the ER and solution behaviors in vitro. MAbs 6:1518–1532. doi:10.4161/mabs.36242

    Article  PubMed  PubMed Central  Google Scholar 

  79. Marotta NP, Lin YH, Lewis YE et al (2015) O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson’s disease. Nat Chem 7:913–920. doi:10.1038/nchem.2361

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Peroutka RJ, Elshourbagy N, Piech T, Butt TR (2008) Enhanced protein expression in mammalian cells using engineered SUMO fusions: secreted phospholipase A2. Protein Sci 17:1586–1595. doi:10.1110/ps.035576.108.of

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Kaufmann H, Fussenegger M (2003) Metabolic engineering of mammalian cells for higher protein yield. New Compr Biochem 38:457–469

    Article  CAS  Google Scholar 

  82. Inducible Protein Expression - T-REx™ System. http://www.thermofisher.com/us/en/home/references/protocols/proteins-expression-isolation-and-analysis/protein-expression-protocol/inducible-protein-expression-using-the-trex-system.html.

  83. Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A 89:5547–5551. doi:10.1073/pnas.89.12.5547

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Gossen M, Freundlieb S, Bender G et al (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268:1766–1769. doi:10.1126/science.7792603

    Article  CAS  PubMed  Google Scholar 

  85. Zhang Y, Katakura Y, Ohashi H, Shirahata S (1997) Efficient and inducible production of human interleukin 6 in Chinese hamster ovary cells using a novel expression system. Cytotechnology 25:53–60

    Article  PubMed  PubMed Central  Google Scholar 

  86. Fussenegger M, Bailey JE, Hauser H, Mueller PP (1999) Genetic optimization of recombinant glycoprotein production by mammalian cells. Trends Biotechnol 17:35–42. doi:10.1016/S0167-7799(98)01248-7

    Article  CAS  PubMed  Google Scholar 

  87. Bi JX, Shuttleworth J, Al-Rubeai M (2004) Uncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1-arrested CHO cells. Biotechnol Bioeng 85:741–749. doi:10.1002/bit.20025

    Article  CAS  PubMed  Google Scholar 

  88. Sinacore MS, Drapeau D, Adamson SR (2000) Adaptation of mammalian cells to growth in serum-free media. Mol Biotechnol 15:249–257. doi:10.1385/MB:15:3:249

    Article  CAS  PubMed  Google Scholar 

  89. Costa AR, Rodrigues ME, Henriques M et al (2011) Strategies for adaptation of mAb-producing CHO cells to serum-free medium. BMC Proc 5(Suppl 8):P112. doi:10.1186/1753-6561-5-S8-P112

    Article  PubMed  PubMed Central  Google Scholar 

  90. Langer ES (2011) Trends in perfusion bioreactors. Bioprocess Int 9:18–22

    Google Scholar 

  91. Li L, Shi M, Song Y et al (2009) A single-use, scalable perfusion bioreactor system. Bioprocess Int:46–54

    Google Scholar 

  92. Kompala DS, Ozturk SS (2006) Optimization of high cell density perfusion bioreactors. Cell culture technologies for pharmaceutical and cell-based therapies. CRC Press, Taylor and Francis Group, Boca Raton

    Google Scholar 

  93. Kolind MP, Nørby PL, Berchtold MW, Johnsen LB (2011) Optimisation of the factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction. J Biotechnol 151:357–362. doi:10.1016/j.jbiotec.2010.12.019

    Article  CAS  PubMed  Google Scholar 

  94. Kaufman RJ, Wasley LC, Davies MV et al (1989) Effect of von Willebrand factor coexpression on the synthesis and secretion of factor VIII in Chinese hamster ovary cells. Mol Cell Biol 9:1233–1242. doi:10.1128/MCB.9.3.1233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Spiegel PC, Kaiser SM, Simon JA, Stoddard BL (2004) Disruption of protein-membrane binding and identification of small-molecule inhibitors of coagulation factor VIII. Chem Biol 11:1413–1422. doi:10.1016/j.chembiol.2004.08.006

    Article  CAS  PubMed  Google Scholar 

  96. Trummer E, Fauland K, Seidinger S et al (2006) Process parameter shifting: Part I. Effect of DOT, pH, and temperature on the performance of Epo-Fc expressing CHO cells cultivated in controlled batch bioreactors. Biotechnol Bioeng 94:1033–1044. doi:10.1002/bit

    Article  CAS  PubMed  Google Scholar 

  97. Zhu MM, Goyal A, Rank DL et al (2005) Effects of elevated pCO 2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: a case study. Biotechnol Prog 21:70–77

    Article  PubMed  Google Scholar 

  98. Yoon SK, Choi SL, Song JY, Lee GM (2005) Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and 37.0 degrees C. Biotechnol Bioeng 89:345–356. doi:10.1002/bit.20353

    Article  CAS  PubMed  Google Scholar 

  99. Mandenius CF, Brundin A (2008) Bioprocess optimization using design-of-experiments methodology. Biotechnol Prog 24:1191–1203. doi:10.1002/btpr.67

    Article  CAS  PubMed  Google Scholar 

  100. Jiang Z, Droms K, Geng Z et al (2012) Fed-batch cell culture process optimization. Bioprocess Int 10:40–45

    CAS  Google Scholar 

  101. Sunley K, Butler M (2010) Strategies for the enhancement of recombinant protein production from mammalian cells by growth arrest. Biotechnol Adv 28:385–394. doi:10.1016/j.biotechadv.2010.02.003

    Article  CAS  PubMed  Google Scholar 

  102. Min Lee G, Koo J (2010) Osmolarity effects, Chinese hamster ovary cell culture. In: Encyclopedia of Industrial Biotechnology. John Wiley & Sons, Inc., pp 1–8

    Google Scholar 

  103. Du Z, Treiber D, McCarter JD et al (2015) Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures. Biotechnol Bioeng 112:141–155. doi:10.1002/bit.25332

    Article  CAS  PubMed  Google Scholar 

  104. Ganne V, Mignot G (1991) Application of statistical design of experiments to the optimization of factor VIII expression by CHO cells. Cytotechnology 6:233–240. doi:10.1007/BF00624762

    Article  CAS  PubMed  Google Scholar 

  105. Torkashvand F, Vaziri B, Maleknia S et al (2015) Designed amino acid feed in improvement of production and quality targets of a therapeutic monoclonal antibody. PLoS One 10:e0140597. doi:10.1371/journal.pone.0140597

    Article  PubMed  PubMed Central  Google Scholar 

  106. Ellert A, Vikström C (2014) Design of experiments with small-scale bioreactor systems for efficient bioprocess development and optimization. Bioprocess Int 12(5):10–13

    Google Scholar 

  107. Pertea M, Lin X, Salzberg S (2001) GeneSplicer: a new computational method for splice site prediction. Nucleic Acids Res 29:1185–1190. doi:10.1093/nar/29.5.1185

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Brunak S, Engelbrecht J, Knudsen S (1991) Prediction of human mRNA donor and acceptor sites from the DNA sequence. J Mol Biol 220:49–65. doi: 0022-2836(91)90380-O [pii]

    Article  CAS  PubMed  Google Scholar 

  109. Solovyev VV, Salamov AA, Lawrence CB (1994) Predicting internal exons by oligonucleotide composition and discriminant analysis of spliceable open reading frames. Nucleic Acids Res 22:5156–5163. doi:10.1093/nar/22.24.5156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Reese MG, Eeckman FH, Kulp D, Haussler D (1997) Improved splice site detection in genie. J Comput Biol 4:311–323. doi:10.1089/cmb.1997.4.311

    Article  CAS  PubMed  Google Scholar 

  111. Rogozin IB, Milanesi L (1997) Analysis of donor splice sites in different eukaryotic organisms. J Mol Evol 45:50–59. doi:10.1007/PL00006200

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biogen Inc., 125 Broadway, Cambridge, MA, 02141, USA

    Christina S. Alves & Terrence M. Dobrowsky

Authors
  1. Christina S. Alves
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Terrence M. Dobrowsky
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christina S. Alves .

Editor information

Editors and Affiliations

  1. National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland

    Paula Meleady

Rights and permissions

Reprints and permissions

Copyright information

© 2017 Springer Science+Business Media LLC

About this protocol

Cite this protocol

Alves, C.S., Dobrowsky, T.M. (2017). Strategies and Considerations for Improving Expression of “Difficult to Express” Proteins in CHO Cells. In: Meleady, P. (eds) Heterologous Protein Production in CHO Cells. Methods in Molecular Biology, vol 1603. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6972-2_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-6972-2_1

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6971-5

  • Online ISBN: 978-1-4939-6972-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation