Assurance of clonality

From a regulatory (FDA) perspective of developing stable cell lines for biotherapeutics, the whole well image is fundamentally important as assurance of clonality.

With the advent of high quality automated imaging in the last 5 years, many customers now only perform a single round of cloning which significantly reduces their timelines for the whole cell line development process (read Systimmune case study)

The numbers of plates screened can vary enormously between customers from a handful to several hundred per project. Part of this can be related to the nature of insertion (random versus targeted) and the efficiency of the seeding methods.

After seeding single cells into 96 well plates, cells are allowed to settle to the bottom of the well or can be centrifuged.

Cells are then imaged by the Cell Metric which is a dedicated imager for assurance of clonality.

The regulator has set out the expectations for the imaging and that it should be carried out on a whole well basis and should be done on Day 0 and then subsequent days of growth and division.

Imaging is carried out for Day 0 and subsequent days of growth and ultimately colony formation. This can be 10-21 days depending on cell types.

A library of images is created for each well and it is possible to track back in time from the colony all the way back to Day 0 and confirm if it started from 1 cell or not (see images below).

Cell Metric Colony

Cell Metric 2 Cells

Cell Metric 1 Cells

For the clonal wells of choice, the Cell Metric can write a hit picking list which can be exported to a third-party liquid handling robot for re-compiling into full 96 well plates of clones.

Finally, a report can be rapidly generated for the well history of the top clones. This documentation package is called the Clonality Report and is importantly something which customers can include as part of their IND submission to the regulator to support their clonality claims.

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