Not Just for Manufacturing
The value of cell-line optimization is not limited to biomanufacturing. For example Cellectis is involved in improving cells for research and gene therapy as well as production. The company’s lead technology is based on meganucleases, or “DNA scissors” that selectively remove genes. Meganucleases have been used for at least 25 years, but Cellectis claims to have engineered these enzymes for greater specificity.
Meganucleases, which are found primarily in single-celled organisms, algae, and some plant organelles, cut chromosomes at specific locations. Despite their relative rarity in nature, meganucleases may be inserted into numerous cell types, where they dependably “cut-and-paste” at target sequences provided the genome contains a specific, 12–30 base pair target sequence. Long recognition sequences are the key to the enzymes’ specificity. If the sequence does not exist naturally it must first be introduced, which has been the major limitation of natural meganucleases.
Cellectis has discovered how to adapt these enzymes to a much broader range of DNA sequences by modifying the meganuclease recognition capability and selecting specific enzymes through high-throughput screening.
The company has thus far worked on workhorse mammalian cells such as HEK293 (human), NIH333 (mouse), and CHO-k1 (hamster), which together cover major areas of interest to both researchers and biomanufacturers. “Clients use our products mostly for drug screening, protein production, and functional genomics,” explains Marc Le Bozec, CEO. Cellectis has enjoyed a “100 percent success rate,” according to Le Bozec, in deleting and inserting genes to produce stable clones.
Cellectis sells kits known as meganuclease recombination systems consisting of specific meganucleases paired with a target DNA template sequence. Users, says Le Bozec, have been 80% successful in targeting specific sites on the genome for gene deletion, insertion, or replacement.
“Each kit provides ten experiments, of which eight should produce a stable, desired clone within four weeks. Moreover it will generate a single copy of a single gene precisely where the customer desires it.” This provides the opportunity, for example, to quantify the activity of two genes located in the same genetic environment. “You can now compare apples to apples,” Le Bozec explains.
By the end of the year the company hopes to introduce kits for knocking down genes by insertion of siRNA. It is planning a next-generation product in 2011 for gene knockout.
Combination of Factors
Rapid gene-splicing techniques notwithstanding, cell-line development takes time in the real world and involves much more than simply introducing genes and firing up a bioreactor. For example Xoma has adapted CHO-k1 cells, a standard production line, to suspension growth and animal component-free media, which “allows more rapid transfer from cell development into process development,” according to Arnie Horwitz, Ph.D., senior director for expression technologies.
During cell-line development Xoma relies on proprietary expression vectors that achieve high expression of the desired gene without conventional gene amplification. Dr. Horwitz calls these “multigenic” vectors because they each contain more than one copy of the desired gene. “This approach increases gene copy number in stable cell lines while eliminating the need for gene amplification.”
Xoma’s cell-line work is complimented by media and feed development efforts that begin with a platform process. The goal is to maintain cells at high density for 14 days or longer to maximize productivity while maintaining product quality. Glycosylation and structural changes are watched closely through “sophisticated analytical tools” that have become part of Xoma’s cell-line selection and optimization process.
Success, Dr. Horwitz says, depends on close interaction between development groups upstream and downstream from cell-line development. “This occurs easily at Xoma because we’re small and able to maintain fluid communication between groups.”
Once cells with the right attributes emerge, they are transferred to separate groups that evaluate them in bioreactors. Quality assessment comes into play here as well, since higher-producing cells may not be the ones that produce product with desirable quality attributes.
“The question of assessing value to cell-line and process development comes up quite often in meetings,” Dr. Horwitz notes. “Both functions are critical. Without transfection, without stable cells possessing acceptable specific productivity you have nothing—everything you do downstream of that becomes more difficult.”
The nature vs. nurture conundrum in cells parallels the evolution of biomanufacturing itself. Biotech was first interested in expression vectors, but for the last 15 years investigators have become more concerned with process optimization. Today, Dr. Horwitz says, the focus is on arriving at a suitable cell line quickly. Hence the interest in rapid transfection and high-throughput clone-selection techniques such as ClonePix FL from Genetix, which claims to reduce selection time by more than 60%.