Genomic rearrangements in CHO producer cell lines

Background.

Currently, nearly 70% of all recombinant protein therapeutics produced worldwide are expressed by Chinese Hamster Ovary (CHO) cells. The adaptability of CHO cells, the ease of their genomic manipulation and their capacity to produce high quality human-like glycosylated proteins largely contributed to this success.

Still, the establishment of producer cell lines is slow-going, and also process development requires time for optimisation as subclones differ in their behaviour. Many subclones need to be tested and frequently the most promising ones turn out to be unstable. Apart from specific silencing of the recombinant promoters, one possible cause for these drawbacks is the high genomic instability of CHO cells: chromosomal rearrangements occur in these cells at a higher frequency than in other comparable immortalised cell lines. While this might be the cause of their easy adaption to different culture conditions and the occurrence of extraordinary high producers, it is also the cause for the major screening efforts required and the need to test a large number of cells. Moreover, advantageous properties are frequently lost in subsequent screening steps and the stability of established producer cell lines is reduced.

Aims and methods.

Apart from karyotyping and FISH analysis of chromosomal regions to test for large scale chromosomal rearrangements, no method is currently described for CHO cells to analyse the extent of genomic changes in CHO cells with time in culture. We are working in an adaptation of a method used in plant breeding and cancer research (Kageyama et al., 2008; Suzuki et al., 2006) for this purpose: Amplified Fragment Length Polymorphism (AFLP).

The method (Meudt and Clarke, 2007) defines an initial pattern of bands of restriction digested genomic DNA and then enables the detection and quantification of changes in this pattern over time in culture. It also allows the characterization of the degree of genomic differences between CHO strains and subclones.
The method can be performed with standard equipment and is medium throughput, so that it can be used on a routine basis for multiple strains and subclones. In a method called dye dilution, cells are stained with a fluorescence dye, which is lost according to the specific growth rate of the cell. It has been observed that subpopulations with different growth rates arise from a uniformly stained starter culture. These subpopulations will be separated by fluorescence analysed cell sorting (FACS) and the different mRNA and miRNA expression patterns will be analysed by microarrays to find out why these subpopulations emerge. The results will be used to find additional candidates which can improve the growth characteristics.

Kageyama et al. (2008) Fluorescence-labeled Methylation-sensitive Amplified Fragment Length Polymorphism (FL-MS-AFLP) Analysis for Quantitative Determination of DNA Methylation and Demethylation Status. Japanese Journal of Clinical Oncology 38(4): 317–322.
Meudt and Clarke (2007) Almost Forgotten or Latest Practice? AFLP applications, analyses and advances. TRENDS in Plant Science 12(3): 1360-1385.
Suzuki et al. (2006) Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage. Cancer Cell 9:199–207.