The author describes expression technology that produces cell lines with high genetic stability. This technology can be used
in multiple cell types using a single or multiple gene constructs.
Genetic and expression stability are important metrics that should be used to evaluate any cell line development method. A
cell line that is genetically stable throughout the biopharmaceutical manufacturing process is essential for any development
program and is required for regulatory adherence. The definition of genetic stability can vary slightly depending on application;
however, typically, genetic stability is confirmation that the transgene DNA and subsequent mRNA sequence along with the number
of transgene copies do not change over the length of time required to perform a biopharmaceutical manufacturing run. The starting
point for genetic stability testing is usually a vial of cells from a research cell bank, master cell bank or working cell
bank. These cells are thawed and cultured for multiple generations. The minimum length of the standard stability study should
be based on maximum number of cell doublings that will occur in the biomanufacturing program. Additional generations can be
added to the study to allow for future changes to the manufacturing process and to be certain that the number of generations
chosen is easily above the maximum doublings possible in the manufacturing run.
The majority of cell line development technologies that result in consistently high biopharmaceutical production generate
cell lines that contain multiple copies of the transgene of interest. Most of these methodologies are based on transfection
of a DNA construct into the desired cell line. The transfected DNA constructs contain a selectable marker, and cell lines
that survive when placed under the selection pressure usually contain the transgene of interest inserted into their genome.
Cell lines produced via transfection of DNA most often contain multiple copy transgene inserts at a single genetic locus. These multi-copy insertion
sites are normally found with the transgenes lined up in a "head to tail" manner (1). Cell lines containing head to tail gene
arrays tend to have problems with genetic stability. During cell mitosis, homologous recombination may occur at the locus
between different sequences within the array. Recombination can cause a reduction in the number of gene copies at the site
and a subsequent decrease in the quantity of mRNA and protein produced. These problems can be accentuated when some form of
gene amplification is performed on the cell line to increase transgene copy number. Amplification methods used during cell
line development typically force the duplication of the head to tail gene array at that same locus in the host cell genome.
The resulting larger array becomes more unstable and more prone to homologous recombination or other replication errors during
cell division. An example of this phenomenon is an antibody-expressing cell line that had been produced using an amplification
method. A significant reduction in the amount of light chain mRNA over extended culture was observed (2). In addition, without
continual selective pressure, dramatic decreases in the number of transgene copies and a subsequent decrease in mRNA levels
were recorded. Major stability differences between clones produced by amplification methodology have been observed (3, 4).
Biopharmaceutical development companies have continually focused on shortening the timeline to get product into clinical trials.
A main roadblock to a shorter timeline is the need to perform a genetic stability study. Even a minimal study (i. e., 40 generations)
takes 3 to 4 months to complete. This timeframe represents a large portion of the cell line development timeline. However,
for cell lines containing multiple gene copies at a single genetic locus, The study must be performed on numerous cell clones
in order to select stable clones to move forward in the development process. The ability to eliminate stability studies from
the critical development path can provide a significant time savings to drug developers.