Removal of fluorescence screening of clones in cell line development: a paradigm shift
Dr Ian Taylor, Chief Commercial Officer, Solentim, blogs on his views following the recent ESACT conference in Copenhagen.
Having just returned from ESACT in Copenhagen, I was impressed once again with the extremely high quality of the poster sessions throughout the event, presented by top level scientists working in labs around the world.
One aspect that surprised me however, and stimulated me for the theme of this blog, were the number of posters which described using fluorescence in their research, both to test clone isolation and to screen clones for potential protein secretion in cell line development.
There is undeniably still a sense amongst a good number of those involved in cell line development that, following transfection, it is necessary to screen thousands of clones in order to identify the small subset which are the high producers. The principle is that using fluorescence detection, either with a labelled antibody or peptide, that it is possible to detect, and subsequently isolate, the cells producing the highest amount of antibody (or any product) very early at the single cell stage. This method is based on a typical clonal distribution for random insertion cloning as shown below (fig 1). On this graph, high producers represent only a small subset of the population, as denoted in the green box.
The concept of screening out “gold nugget” high producers with fluorescence is not new; whilst working at Genetix back in 2005, myself and colleagues developed the ClonePix FL for precisely this purpose. The ClonePix FL enabled cells to be isolated in semi-solid media, then as these grew and secreted antibody into the media, a fluorescently labelled secondary antibody was used to detect and “pick out” high producing clones.
However, it became apparent that there was a massive flaw in this approach, and this same flaw exists for current methods using fluorescence detection. Namely, there is a lack of correlation between fluorescence at the single cell stage compared with the eventual productivity for expanded clones in shaking media. In reality, the fluorescence screen simply eliminates the population of non-producing clones (those not fluorescing in the screen) rather than identifying high producers. This makes instruments such as the Cytena f.sightⓇ, Cyto-MineⓇ and to an extent the BeaconⓇ, expensive systems that isolate fluorescent clones, revealing little to nothing about the true productivity ranking of the clones when grown in shaking culture.
In my opinion, it is now crucial to ‘bang the drum’ for a different approach which changes the dynamics of the cell line development workflow. This approach is to use transposases. By using clever vector design and targeted gene integration, it is possible to create a population whereby the majority of clones have the GOI inserted into specific sites, generating clones with high, predictable expression and stability. Some Pharma companies, such as Pfizer, have their own proprietary approaches using vectors containing ‘landing pads’ to achieve this. However, transposases offer an excellent commercial alternative.
Solentim has recently collaborated with ATUM– one such company providing transposases (Leap-in™). The game changer here is that transposases shift the clonal distribution massively towards high producing clones; for example, in Figure 2 below, 62% of clones are shown to be in the first quartile for productivity.
In practical terms, this entirely removes any rationale for fluorescence, as the majority of clones from transfection will inherently be high producers. Therefore, the only requirement for successful cell line development is an efficient, reliable single cell isolation and documentation system (e.g. the VIPS). An additional benefit of this transposase approach is that clones also exhibit high inherent stability, therefore taking stability studies off the critical path (saving even more time).
Further endorsement of the impact of transposases has been demonstrated in Lonza’s recent announcements recommending the GS piggyBac™ transposases.
The take-home message here for cell line development groups is to understand that fluorescence screening is an outdated approach, providing a binary result of producer vs non-producer. Contrastingly, using transposases is quick, easy and only requires a handful of plates for a complete project. Following this, the clones can be taken to shaking media to confirm the highest expressors.