Standard clone-selection methodology involving limited-dilution cloning is a time-consuming, hit-or-miss process that often requires six months of intense labor to generate small cultures of a chosen clone. We have used a new clone-selection technology called Cell Xpress to isolate high-producing CHO cell clones.
The Cell Xpress clone-selection technology is a combination of high-speed image scanning and quantitation with high-speed laser manipulation that allows for in situ measurement of recombinant protein secretion on a single cell basis followed by laser-mediated destruction of the low-producing cells in a population.
Cell Xpress utilizes the LEAP™ (Laser-Enabled Analysis and Processing) platform and incorporates an immunofluorescence-based assay for identifying the highest secreting clones in either transfected or stable pools of cells. Cells are seeded into 384-well tissue culture plates in the presence of a matrix designed to capture secreted IgG molecules.
After overnight incubation, captured IgG is detected via a secondary reagent containing a conjugated fluorophore, and the viable cells are stained with live cell tracking dye. Following a short incubation, the captured IgG secretions and associated live cells are visualized using the LEAP instrument. The Cell Xpress software can then be used to identify and retain the highest producing cells in the population for cloning and expansion while removing the undesired cells from the population by laser-induced apoptosis.
The Cell Xpress system can rapidly screen and process a 384-well plate with 150–200 cells/well in less than one hour making it possible to evaluate over 40,000 individual cells for selecting the highest secreting clones (Figure 1A). In the current study, eight clones were selected from two separate 384-well plates and expanded to spinner flask cultures (60-mL volume) within a 10–12 week period.
Of the eight clones generated, five exhibited increased specific productivity (qP) ranging from 15–40 pg/cell/day compared to the initial cell line that had a specific productivity of 10–12 pg/cell/day under identical growth conditions. Four of these clones (Figure 1B) were chosen for media screening to identify the preferred clone for process development.