Cell line development is a critical part of biopharmaceutical production. The aim is to create a line that expresses the desired protein efficiently and consistently. But although current methods work, they are complex and time consuming, says Yaakov Benenson, PhD, at ETH Zurich in Switzerland.

“The two most important features of an optimal producer cell line, stability and high productivity, often pull in opposite directions, he explains. “The conventional workflow for cell line development proceeds as follows: a transgene encoding the protein-of-choice is knocked into the cell’s genome at an unknown location, a.k.a. random integration; transgene copy number is expanded with the use of selection drugs to increase product titers; and hundreds or thousands of cell line candidates thus produced are screened until a few stable, high producers are found.”

This random integration step is a problem, continues Benenson, because it means developers need to search for the clone in which insertion has taken place in the best place in the genome.

“Selection is long and laborious because random integration and drug-based gene amplification make cells intrinsically unstable,” he says.

To address this, Benenson and his colleague, Raffaele Altamura, PhD, developed a means of inserting the protein-encoding transgene at a specific point, details of which were published in a recent study in Nucleic Acids Research.

“Our cell line development strategy is designed to build a biomanufacturing `island’ within the genome of a cell leaving nothing to chance. The number and exact location of the genetic elements and transgenes needed to express a therapeutic protein are fully specified, and every single nucleotide is known,” he tells GEN. “Biomanufacturing islands are designed to be stable by design, and transgene copy number and integration sites are known from the outset. The key innovation is diversifying the genetic sequence of the protein-coding gene itself without changing the protein, as well as of the gene regulatory sequences.”

This approach lets researchers put together cassettes with multiple gene copies of the protein-coding gene of interest (10 or more) that can be integrated into a specified site in a CHO cell line.


To date the approach has not been used commercially. However, Altamura predicts the specificity and precision of the technique will be of interest to industry.

“The key objective is to shorten the cell line development timeline to as few as four to six weeks, as opposed to the six months-plus often required to establish a producer cell line with the conventional, amplification-and-selection workflow,” he says. “All this while ensuring cell line stability, as well as predictable and satisfactory product titers.”

According to Altamura, “The approach would not require new technology beyond our published software tool. From the practical standpoint, it requires the infrastructure and know-how for implementing yeast assembly via homologous recombination. This is itself an established technology, but it does require specialized knowledge.”