Transfection and enrichment

Process map

The initial goal of cell line development is to identify rare, stable, monoclonal cells secreting high titres of a biologically active recombinant protein such as a monoclonal antibody (mAb).

Many different strategies can be used to produce a pool of recombinant cells, with different laboratories preferring or specialising in different transfection methods. The standard approach starts with a bulk transfection (around 10-20 x 106 cells) of cells such as CHO-S, followed by clone selection, typically with methotrexate (MTX) amplification or Lonza’s glutamine synthetase (GS) system. However, the random integration of recombinant DNA is an inefficient way of generating high-producing clones. It is therefore necessary to screen many hundreds or thousands of transfected cells to isolate a clone with suitable properties.

Some newer approaches in cell line engineering such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and meganucleases have been designed to target transgene integration to specific DNA sequences in the genome. This can increase the frequency of stable high-producers and reduce the number of clones for screening.

After transfection or fusion, certain fluorescence-based methods can be used to enrich for high producers, for example:

  • FACS-based methods such as cold capture, affinity matrix attachment and gel microdrop assays
  • culture of clones in semi-solid medium, followed by detection of secreted recombinant proteins as halo fluorescence and robotic or manual picking of the most productive clones (clones then have to re-adapt to liquid culture; note that the level of productivity in semi-solid media does not always correlate well with the level of productivity in liquid culture)

The next step

The top candidate clones are seeded into microplates for further investigation. For more information, see cell seeding.

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