Cloning Method Qualification

As timelines for the development of cell lines producing important biologics is extremely tight, ensuring cloning methods are validated and producing consistent results is of high importance for cell line development groups, as well as for downstream groups that are reliant upon the Master Cell Bank consistently producing stable quality product.

The Cell Metric range offers users the ability to verify clonality using bright field as standard, however, with the introduction of fluorescence (Red and Green object rare event detection), users are now able to use this feature to rapidly qualify their cloning method.

Mixed Population Method Validation:

The system’s fluorescence features were design to provide the user with a rapid overview of the cloning method performance via the plate map, as well as present data for export for further cloning method data analysis.

The Cell Metric continues to use bright field imaging but will image biology in both red and green fluorescence channels also.

The workflow and steps below outline how the Cell Metric with its rare event fluorescence detection feature can be used to validate the entire cloning method (Cell Culture, Cell Preparation, Cell Deposition, Settling methods prevalence of ghost wells etc)

 

Workflow illustration

 

Step 1:

The cell line of interest is prepared by dividing the population into two and then fluorescently labelling the cell populations via cell staining or transfecting them with fluorescent proteins (RFP/GFP). The cell populations are then mixed and can be cultured together, if desired, to encourage cell to cell interactions.

Step 2:

The mixed population is then single cell deposited via the method of choice; FACS, Limiting dilution or other methods, and allowed to settle to the bottom of the plate via centrifugation or by leaving the plates to passively settle for set period of time.

Step 3:

The plates are then imaged on the Cell Metric using the Cloning Method Qualification Application at Day 0 and any subsequent days as required.

Step 4:

The Cell Metric will display the detected rare events per well in a plate format for quick review of the cloning method performance. This data can be interrogated and exported for further review and analysis.

Single Population Method Quality Control:

As with validating the cloning method used for the generation of clonality derived cell lines, it is important to ensure that the method does not deviate and continues to perform as expected. For example, with limiting dilution, there could be a higher prevalence of empty wells if the cell culture wasn’t prepared as previously optimised, or with FACS, there could be a higher prevalence of doublets if the settings are not as originally optimised. If regular QC checks are carried out, any erroneous results will be noticed and changes can be made before allowing the cells to grow into clones.

In this method, cells are labelled with a single fluorescence colour and detected in a single fluorescence channel and bright field.

The workflow in the application note outlines how regular QC checks of the defined seeding method can be carried out using the fluorescence option; either in parallel to the standard seeding experiment (bright field only) or prior to the seeding experiment to allow for any changes that needs to be carried out.

Method quality control illustration