While cell-service companies may argue persuasively for their pet technologies, cell-line optimization can proceed from several different angles.
Andrew Sandford, vp of business development at Selexis, describes his company as a “pure-play developer of technologies for rapidly producing stable, high-performing cell lines for bioproduction.”
The company’s rapid cell-line development technology, Selexis Genetic Elements™ (SGEs), are human DNA sequences that control the dynamic organization of transgenes in the chromatin. SGEs insulate nearby genes from the effect of surrounding chromatin, thereby increasing copy-number-dependent, position-independent gene expression. From a cell-line development perspective, SGEs increase the number of independently transformed cells that express the protein of interest, and promote higher levels of protein expression.
SGEs, which are found only in higher organisms, also possess regulatory functions that control the expression of many genes at a time. “There are thousands of these elements in the genome,” says Pierre-Alain Girod, Ph.D., CSO. Not all elements work in every cell line or species. Selexis has tested SGEs that are optimal for CHO, HEK (human embryonic kidney), CAP (Cevec amniocyte production), and other biomanufacturing-worthy cells.
“SGEs boost not only the number of copies of DNA integrated into the genomes of mammalian cells, but the quantity of messenger RNA generated, and makes the integration sites more stable to long-term expression,” Sandford explains. “Because of how these elements work, we can condense development time to 10 to 12 weeks post-transfection, from gene to a stable clonal production cell line.” Selexis claims titers of between three and five g/L for optimal Chinese hamster ovary (CHO) cell lines, and between one and three g/L for their “bread and butter” unoptimized batch processes.
In addition to its cell-line development and gene-expression platforms, Selexis employs a cell culture platform based on high-performing CHO cells that thrive in basal media and a specially designed feed regimen. Cell development and culture components must be synchronized, Sandford says, to achieve highly stable, productive cell lines in three months.
After transfection, Selexis uses antibiotics to kill off cells that do not carry the desired gene and limits dilution cloning to isolate cells. “Instead of trying to find a needle in a haystack, we have a stack of needles to choose from,” Sandford explains. “Not needing to engage in mechanical separation to isolate high-performing cells is a tremendous advantage from the perspective of throughput. We’re not limited by the number of flow cytometry systems we have.”
Lonza creates high-producing cell lines and processes for cells developed by customers as part of its comprehensive biopharmaceutical development services. Lonza’s cell-related services include vector construction, cell-line construction (transfection and cloning), cell-line evaluation in shake-flasks and bioreactors, cell bank creation and characterization, bioreactor process optimization, and bioreactor process characterization.
Lonza recently introduced a new cell-line construction process that transfects and clones in the same step. The process, which involves direct cloning from transfectant pools using fluorescence-activated cell sorting (FACS), reduces timelines from transfection to cGMP master cell bank creation to about 19 weeks. “As part of this construction, we use a screening process that is relevant to the production process,” says Alison Porter, Ph.D., senior science leader at Lonza’s Slough, U.K. facility. “The screens we use in cell-line creation are therefore designed to partner with our production process.”
The company’s philosophy regarding production is to develop platform inoculum and processes that employ standardized media, feeds, and culture conditions with model cell line(s). These provide speed to clinic while retaining the possibility of further optimization.
Since 1990 Lonza says its product titers have increased 200-fold as a result of improvements in specific production rate, viable cell concentration, and culture time. “Expression system, vector design and selection, and screening all contribute to this,” Dr. Porter notes, “but the major influencers of product concentration have been process conditions and the cell line, and of the former the design of media, and feeds in particular, have been of most importance.”
Moving forward, she believes that host cell-line engineering will likely result in further advances in product concentration, for example by controlling apoptosis and/or assisting translation and product secretion.