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Related Concept Videos

Hybridoma Technology01:31

Hybridoma Technology

Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or epitope. Such antibodies are used in research, diagnostics, and disease therapy. The hybridoma technology established in 1975 by Georges Köhler and Cesar Milstein was awarded the Nobel Prize in Medicine in 1984 for revolutionizing research and therapy.
Hybridoma Selection
Commonly used fusion techniques — electroporation, polyethylene glycol...

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Laboratory Scale Production and Purification of a Therapeutic Antibody
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Laboratory Scale Production and Purification of a Therapeutic Antibody

Published on: January 24, 2017

An efficient and targeted gene integration system for high-level antibody expression.

Ying Huang1, Yan Li, Yu Gang Wang

  • 1Department of Molecular Immunology, Beijing Institute of Basic Medical Sciences, Beijing 100850, PR China.

Journal of Immunological Methods
|March 14, 2007
PubMed
Summary

This study introduces a novel FRT/FLP system for stable, high-level recombinant antibody expression in cell lines, overcoming unpredictable position effects common in random integration methods. The new system enables precise targeting and amplification of antibody genes for efficient production.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Cell Line Engineering

Background:

  • Random integration for recombinant antibody production is laborious and results in unpredictable expression levels due to genomic position effects.
  • Existing methods lack precise control over gene integration, limiting stable and high-level expression of therapeutic antibodies.

Purpose of the Study:

  • To develop a cell-vector system utilizing the FRT/FLP strategy for site-specific integration and high-level antibody expression, mitigating position effects.
  • To identify and characterize chromosomal loci suitable for stable gene amplification and high transcriptional activity.

Main Methods:

  • Constructed a plasmid with dual weakened markers (galactosidase and dihydrofolate reductase, DHFR) and an FRT sequence for screening suitable host cell loci.
  • Transfected Chinese hamster ovary (CHO) cells and selected candidate lines using the markers.
  • Developed an antibody gene-targeting vector with an FRT-fused hygromycin gene for site-specific integration via FLP recombinase.
  • Utilized Southern blot and fluorescence in situ hybridization (FISH) for genomic analysis and locus characterization.

Main Results:

  • Identified 20 candidate cell lines from 721 clones exhibiting desired transcriptional and amplification characteristics.
  • Confirmed site-specific integration of antibody genes into the targeted FRT locus in engineered cell lines.
  • Achieved high-level expression of three recombinant antibodies in a selected cell line (number 37), with the highest producer yielding over 200 µg/ml in 6 days.

Conclusions:

  • The FRT/FLP system effectively overcomes position effects, enabling predictable and high-level recombinant antibody expression.
  • Site-specific integration into engineered loci facilitates stable gene amplification and consistent antibody production.
  • This strategy offers a robust platform for developing high-producing cell lines for biopharmaceutical manufacturing.