Isolation of candidate mammalian cell clones by limiting dilution, ring cloning, or simple manual collection of colonies is a time-consuming, resource-intensive, and costly procedure that is prone to cross contamination of cells and user error. One major disadvantage commonly encountered is the difficulty in establishing monoclonality—an essential step in the production of therapeutic proteins and antibodies.
To overcome many of the drawbacks typically associated with isolating choice mammalian cell clones, Genetix has developed the ClonePix™ FL system for screening and selection of secretory cell lines. The utility of ClonePix FL lies in its ability to visualize and quantify proteins secreted from thousands of clones in situ, and to select and accurately isolate only the highest value secretors, shortening time scales and simplifying downstream culture.
The ClonePix FL solution has been validated for specific detection of secreted antibodies and monomeric proteins, cell surface proteins, and intrinsic GFP fusion proteins. It is compatible with a range of cell types including hybridoma, myeloma, HEK293, and both suspension-adapted and adherent CHO cells.
A fundamental initial step in the workflow (Figure 1) is the culturing of mammalian cell lines in semisolid media, such that cells form discrete colonies, each originating from a single parent cell. Heterogeneous populations of thousands of cells are grown into clonal colonies in either one-well or six-well microplates. The cells are then screened rapidly, using white light to detect the colonies and fluorescence to quantify secreted protein in situ.
The secreted protein-detection assay requires the secreted product to be immobilized around the colony using a fluorescent detection probe. Dedicated ClonePix FL software quantifies the fluorescence associated with each colony and drives the automated picking of only the most desirable clones into 96-well destination plates. Figure 2 shows example images that identify the highest producing clones. A variety of different proteins can be detected and isolated simultaneously by using detection agents labeled with different fluorophores (multiplexing).
For hybridoma fusions, clones can be selected based on antigen-specificity by additionally adding antigen as the detection probe; this can be directly conjugated with fluorescence or via a secondary detection agent. Plates of picked colonies are then grown to confluence in preparation for clone expansion and scale-up.