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The absence of effect of gene copy number and mRNA level on the amount of mAb secretion from mammalian cells

Abstract

Recombinant human antibody production represents a major growing class of biopharmaceuticals based on the technological progress within the last decades especially in CHO cells. The HIV neutralizing human monoclonal antibody 2F5 was developed as hybridoma from human lymphocyte preparations. In order to estimate the potential of recombinant 2F5-expressing CHO cells, we generated different recombinant CHO cell lines by varying regulatory sequences, the codon usage, the signal peptides, and the transfection technique. These 2F5-expressing cell lines were developed by selection of the best producer, clone homogeneity, and clone stability. The gene copy number of the clones differed significantly due to methotrexate amplification. In one cell line, we identified only one copy of heavy chain and two copies of light chain. Neither the gene copy number nor the promoter was found to influence the amount of transcript exclusively emphasizing the positioning effect of the transgene. Messenger RNA levels were highest in 2F5/CO and may have resulted from a combination of the promoter and codon-optimized sequences, but unexpectedly, the amount of secreted product was not elevated in this configuration. In our example, translational and post-translational limitations are responsible for decreased antibody secretion.

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Acknowledgments

This research was part of the Pharma-Planta Project (LSHB-CT-2003–503565), kindly funded by an EU FP6 program and partly supported by Polymun Scientific GmbH.

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Correspondence to Renate Kunert.

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Reisinger, H., Steinfellner, W., Stern, B. et al. 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 (2008). https://doi.org/10.1007/s00253-008-1701-1

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Keywords

  • CHO cells
  • GCN
  • Monoclonal antibody
  • qPCR
  • Protein-free transfection
  • Signal peptide