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Cytotechnology

, Volume 63, Issue 4, pp 351–362 | Cite as

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

  • Y. PrietoEmail author
  • L. Rojas
  • L. Hinojosa
  • I. González
  • D. Aguiar
  • K. de la Luz
  • A. Castillo
  • R. Pérez
Original Research

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.

Keywords

Recombinant antibody Protein expression stability NS0 myeloma cell line Proteomics 

Notes

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.

Supplementary material

10616_2011_9348_MOESM1_ESM.tif (166 kb)
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)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Y. Prieto
    • 1
    Email author
  • L. Rojas
    • 1
  • L. Hinojosa
    • 1
  • I. González
    • 1
  • D. Aguiar
    • 1
  • K. de la Luz
    • 1
  • A. Castillo
    • 1
  • R. Pérez
    • 1
  1. 1.Research and Development DirectionCenter of Molecular ImmunologyPlaya HavanaCuba

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