Cloning stable cell lines with high specific protein secretion is challenging, time consuming, and a major bottleneck in biopharmaceutical process development. Optimal production cell lines have high levels of secretion of the target recombinant protein, stable expression of the secreted protein, and growth characteristics indicative of successful transferability to bioreactors.
Traditionally, clone selection using limiting-dilution cloning or colony picking is labor and materials intensive, time consuming, and not amenable to significant automation. Large numbers of presumptive clones must be distributed over many multiwell plates, manually identified and verified as clones, and grown to significant cell numbers for assay before ranking and selection.
Random isolation of clones followed by large-scale assay inherently leads to significant amounts of effort wasted on non-secreting clones. Once the ranking process is completed, a large number of clones must be maintained over time in order to select clones with stable secretion rates. Inherent limits of scale and lack of automation restrict the size of selection pools to 1,000–2,000 cells and preclude multiple parallel projects.
Cyntellect’s CellXpress™ is a fundamentally new process that applies an in situ live-cell fluorescence secretion assay, brightfield imaging, and in situ laser purification to clone selection, clone validation, growth tracking, and secretion stability tracking. CellXpress provides direct, automated, in-plate clone selection using standard growth media within a constantly sterile environment. Cells are not, for example, transferred to a semisolid growth medium as required for semiautomated colony pickers.
CellXpress reduces the time from stable pool to a set of clones ranked on implied specific productivity to less than 24 hours. This substantially reduces the workload upfront, focusing only on clones that have high potential value downstream. Subsequently, clones are assessed and ranked based upon in situ measurement of growth rates and secretion stability permitting early attrition of many clones. CellXpress has been effectively implemented on a number of cell types, including CHO and NS0.
CellXpress is powered by LEAP™, a high-throughput, in situ laser-processing platform, that images the full well of multiwell plates, identifies each cell via fluorescence or brightfield imaging, quantifies fluorescence intensity from each cell, gates cell populations on combinations of cell-level features, and rapidly (>1,000 pulses per second) targets undesired cells for elimination from a well via precisely targeted laser irradiation, while protecting the desired cell to be retained.