A Good Pedigree
While nurture has been responsible for most of the recent improvements in volumetric productivity, starting with a high-producing cell line is of utmost importance. Poorly producing cells can be improved 100-fold and still not be up to production standards. A high producer for which a three- or fourfold improvement is achieved through media/feed/supplement strategies, and another doubling or so during process development, can make a huge difference in subsequent cost of goods and capital expenditures.
“We have never seen a cell line that makes less than 100 mg/L be improved to the grams per liter range using process optimization,” says Simin Zaidi, manager of process development at Avid Bioservices, “especially given schedule restraints most projects have.”
She cites as an example a CHO cell line producing an IgG at 70 mg/L that was improved to 350 mg/L through an extensive optimization effort. Another cell line that began at 400 mg/L with process optimization easily reached 1g/L with only minimal work. “And there is a good chance that this could be improved even further with additional process-development work.”
In addition to production services, Avid provides optimization competencies that fall under both nature and nurture categories. Avid’s cell-line development group optimizes cells, then passes them off to process development, which “works magic with media and feeds,” according to cell line development manager Jeanette Doerr, Ph.D.
Like most early-stage development services companies, Avid uses the standard limiting dilution method for generating clones. Recently the company adopted the ClonePix™ FL cell screening and selection platform from Genetix. Where the dilution method takes about eight labor-intensive weeks to screen 1,000 clones, ClonePix processes 10 times as many cells in about three weeks, according to the company. The instrument provides a visual image, based on fluorescence detection, of the highest-producing colonies, and ultimately individual cells. ClonePix also confirms that a colony is truly clonal, whereas limiting dilution requires examining every single cell.
Another interesting cytometry system for clone and cell line selection is LEAP, a microplate-based cytometry system from Cyntellect that CEO Fred Koller, Ph.D., says satisfies requirements for both nature and nurture. LEAP identifies the highest-producing cells from either genetically heterogeneous collections of cells or, under varying culture conditions, for a stable clone.
LEAP generates feature-rich images of cells in a closed environment, which flow cytometry and bead-based selection methods do not, Dr. Koller says. Instead of picking the most promising cells, LEAP leaves them in place and obliterates the undesirable cells through laser irradiation. This assures that colonies arise from a single cell.
“The cells you choose haven’t been taken anywhere or otherwise manipulated,” notes Dr. Koller. “They are unaware of what has happened to neighboring cells.” Excessive manipulation, he says, can change a cell’s gene expression, particularly for adherent cells. “It’s particularly important not to disturb cells during measurement periods.” Once cells are selected, they may be removed and cultured within larger vessels.