Skip to main content
SpringerLink
Log in

Towards the molecular characterization of the stable producer phenotype of recombinant antibody-producing NS0 myeloma cells

  • Original Research
  • Published:
Cytotechnology Aims and scope Submit manuscript

Abstract

The loss of heterologous protein expression is one of the major problems faced by industrial cell line developers and has been reported by several authors. Therefore, the understanding of the mechanisms involved in the generation of stable and high producer cell lines is a critical issue, especially for those processes based on long term continuous cultures. We characterized two recombinant NS0 myeloma cell lines expressing Nimotuzumab, a humanized anti-human epidermal growth factor receptor (EGFR) antibody. The hR3/H7 clone is a stable producer obtained from the unstable hR3/t16 clone. The unstable clone was characterized by a bimodal distribution of intracellular immunoglobulin staining using flow cytometry. Loss of antibody production was due to the emergence of a non-producer cell subpopulation that increased with cell generation number. Immunoglobulin heavy chain (HC) and light chain (LC) ratio (HC/LC) was lower for the unstable phenotype. Proteomic maps using two dimensional gel electrophoresis (2DE) were obtained for both clones, at initial cell culture time and after 40 generations. Fifteen proteins potentially associated with the phenomenon of production stability were identified. The hR3/H7 stable clone showed an up-regulated expression pattern for most of these proteins. The regulation of recombinant antibody production by the host NS0 myeloma cell line most likely involves simultaneously cellular processes such as DNA transcription, mRNA processing, protein synthesis and folding, vesicular transport, glycolysis and energy production, according to the proteins identified in the present proteomic study.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Agshe VR, Hartl F-U (2000) Roles of molecular chaperones in cytoplasmic protein folding. Cell Dev Biol 11:15–25

    Article  Google Scholar 

  • Alete D, Racher AJ, Birch JR, Stansfield SH, James D, Smales CM (2005) Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities. Proteomics 5:4689–4704

    Article  CAS  Google Scholar 

  • Barnes L, Dickson A (2006) Mammalian cell factories for efficient and stable protein expression. Curr Opin Biotechnol 17:381–386

    Article  CAS  Google Scholar 

  • Barnes L, Bentley C, Dickson A (2000) Advances in animal cell recombinant protein production: GS-NS0 expression system. Cytotechnology 32:109–123

    Article  CAS  Google Scholar 

  • Barnes L, Bentley CM, Dickson A (2003) Stability of protein production from recombinant mammalian cells. Biotechnol Bioeng 81:631–639

    Article  CAS  Google Scholar 

  • Barnes L, Bentley C, Dickson A (2004) Molecular definition of predictive indicators of stable protein expression in recombinant NS0 myeloma cells. Biotechnol Bioeng 85:115–121

    Article  CAS  Google Scholar 

  • Barnes L, Bentley CM, Moy N, Dickson AJ (2007) Molecular analysis of successful cell line selection in transfected GS-NS0 myeloma cells. Biotechnol Bioeng 96:337–348

    Article  CAS  Google Scholar 

  • Castillo A, Victores S, Faife E, Rabasa Y, de la Luz KR (2005) Development of an integrated strategy for recombinant cell line selection. In: Gódia F, Fussenegger M (ed) Animal cell technology meets genomics. Springer, Berlin, pp 505–507

  • Chevalier S, MacDonald N, Tonge R, Rayner S, Rowlinson R, Shaw J, Young J, Davison M, Roberts R (2000) Proteomic analysis of differential protein expression in primary hepatocytes induced by EGF, tumour necrosis factor alpha or the peroxisome proliferators nafenopin. Eur J Biochem 267:4624–4634

    Article  CAS  Google Scholar 

  • de la Luz KR, Rojas L, Victores S, Lage A, Eyers C, Hart S, Castellanos L, Castillo A, Gaskell S (2008) Proteomic analysis of the adaptation of the host NS0 myeloma cell line to a protein-free medium. Biotecnología Aplicada 24:215–223

    Google Scholar 

  • Dinnis D, James D (2005) Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature? Biotechnol Bioeng 91:180–189

    Article  CAS  Google Scholar 

  • Dorai H, Csirke B, Scallon B, Ganguly S (2006) Correlation of heavy and light chain mRNA copy numbers to antibody productivity in mouse myeloma production cell lines. Hybridoma 25:1–9

    Article  CAS  Google Scholar 

  • Faife E, Rodríguez T, Rabaza Y, Badía T, Victores S, Castillo A (2008) Effects of selection and adaptation of NS0 cells to protein-free medium on the properties, affinity and biological activity of a monoclonal antibody. Biotecnología Aplicada 25:247–253

    CAS  Google Scholar 

  • Gharahdaghi F, Weinberg CR, Meagher DA, Imai BS, Mische SM (1999) Mass spectrometric identification of proteins from silver-stained polyacrylamide gels: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20:601–605

    Article  CAS  Google Scholar 

  • Hutchison S, LeBel C, Blanchette M, Chabot B (2002) Distinct sets of adjacent heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 binding sites control 5′ splice site selection in the hnRNP A1 mRNA precursor. J Biol Chem 277:29745–29752

    Article  CAS  Google Scholar 

  • Köhler G, Milstein C (1976) Derivation of specific antibody-producing tissue culture and tumour lines by cell fusion. Eur J Inmunol 6:511–519

    Article  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • MacIver NJ, Jacobs SR, Wieman HL, Wofford JA, Coloff JL, Rathmell JC (2008) Glucose metabolism in lymphocytes is a regulated process with significant effects on immune cell function and survival. J Leukoc Biol 84:949–957

    Article  Google Scholar 

  • Martínez A, Mulshine JL (2002) Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as a marker of preinvasive lung cancer. J Clin Ligand Assay 25:100–103

    Google Scholar 

  • Mateo C, Moreno E, Amour K, Lombardero J, Harris W, Pérez R (1997) Humanization of mouse monoclonal antibody that block the EGF-R: recovery of antagonistic activity. Immunotechnology 3:71–81

    Article  CAS  Google Scholar 

  • Nasim FU, Hutchison S, Cordeau M, Chabot B (2002) High-affinity hnRNP A1-binding sites and duplex–forming inverted repeats have similar effects on 5′ splice site selection in support of a common looping out and repression model. RNA 8:1078–1089

    Article  CAS  Google Scholar 

  • Nogales E, Wolf SG, Downing KH (1998) Structure of the alpha beta tubulin dimer by electron crystallography. Nature 391:199–203

    Article  CAS  Google Scholar 

  • Ou XM, Chen K, Shih JC (2006) Monoamine oxidase A and repressor R1 are involved in apoptotic signaling pathway. Proc Natl Acad Sci USA 103:10923–10928

    Article  CAS  Google Scholar 

  • Piskunova TS, Yurova MN, Ovsyannikov AI, Semenchenko AV, Zabezhinski MA, Popovich IG, Wang ZQ, Anisimov VN (2008) Deficiency in poly (ADP-ribose) polymerase-1 (PARP-1) accelerates aging and spontaneous carcinogenesis in mice. Curr Gerontol Geriatr Res 754190. Epub Apr 14. PMID: 19415146

  • Rojas LE, de la Luz KR, Victores S, Castellanos L, Gaskell S, Castillo A, Pérez R (2005) Comparative proteomic study of mechanisms involved in the expression of recombinant monoclonal antibody in non amplified NS0 myeloma cell line. In: Smith R (ed) Cell technology for cell products. Springer Publisher, Berlin, pp 715–721

  • Schlatter S, Stansfield S, Dinnis DM, Racher A, Birch J, James D (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  Google Scholar 

  • Seth G, Philp RJ, Lau A, Jiun KY, Yap M, Hu WS (2007) Molecular portrait of high productivity in recombinant NS0 cells. Biotechnol Bioeng 97(4):933–951

    Article  CAS  Google Scholar 

  • Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858

    Article  CAS  Google Scholar 

  • Smales CM, Dinnis DM, Stansfield SH, Alete D, Sage EA, Birch JR, Racher AJ, Marshall CT, James DC (2004) Comparative proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate. Biotechnol Bioeng 88:474–488

    Article  CAS  Google Scholar 

  • Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg effect: the metabolic requirements of cell proliferation. Science 324:1029–1033

    Article  CAS  Google Scholar 

  • Voet D, Voet JG (1995) Translation. In: Rose N (ed) Biochemistry, second edition. Wiley, New York, pp 997–1003

  • Wallrath LL (1998) Unfolding the mysteries of heterochromatin. Curr Opin Genet Dev 8:147–153

    Article  CAS  Google Scholar 

  • Whitford W (2003) NS0 serum-free culture and applications. BioProcess Int 12:36–47

    Google Scholar 

  • Wilson C, Bellen HJ, Gehring WJ (1990) Position effects on eukaryotic gene expression. Annu Rev Cell Biol 6:679–714

    Article  CAS  Google Scholar 

  • Wurm FM (2004) Production of recombinant protein therapeutics in cultivated mammalian cell. Nat Biotechnol 22:1393–1398

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. Yassel Ramos Gómez from the Center of Genetic Engineering and Biotechnology (CIGB, Havana, Cuba) for his valuable help in the proteomics assays and Dr. Alejandro López-Requena for a careful reading of the manuscript.

Author information

Authors and Affiliations

  1. Research and Development Direction, Center of Molecular Immunology, PO Box 16040, 216 St. & 15th Ave, Atabey, Playa Havana, 11600, Cuba

    Y. Prieto, L. Rojas, L. Hinojosa, I. González, D. Aguiar, K. de la Luz, A. Castillo & R. Pérez

Authors
  1. Y. Prieto
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. L. Rojas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. L. Hinojosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. I. González
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D. Aguiar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. K. de la Luz
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. A. Castillo
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. R. Pérez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Y. Prieto.

Electronic supplementary material

Below is the link to the electronic supplementary material.

10616_2011_9348_MOESM1_ESM.tif

Supplementary figure 1 . Immunoblot analysis of hR3 antibody chains. HC and LC polypeptides from purified hR3 antibody were detected using an alkaline phosphatase-conjugated anti-human γ-chain antibody or a horseradish peroxidase-conjugated goat anti-human ĸ chain antibody, respectively. (TIFF 166 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Prieto, Y., Rojas, L., Hinojosa, L. et al. Towards the molecular characterization of the stable producer phenotype of recombinant antibody-producing NS0 myeloma cells. Cytotechnology 63, 351–362 (2011). https://doi.org/10.1007/s10616-011-9348-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10616-011-9348-7

Keywords

Search

Navigation