Karyotype variation of CHO host cell lines over time in culture characterized by chromosome counting and chromosome painting.

Genomic rearrangements are a common phenomenon in rapidly growing cell lines such as Chinese hamster ovary (CHO) cells, a feature that in the context of production of biologics may lead to cell line and product instability. Few methods exist to assess such genome wide instability. Here, we use the population distribution of chromosome numbers per cell as well as chromosome painting to quantify the karyotypic variation in several CHO host cell lines. CHO-S, CHO-K1 8 mM glutamine, and CHO-K1 cells adapted to grow in media containing no glutamine were analyzed over up to 6 months in culture. All three cell lines were clearly distinguishable by their chromosome number distribution and by the specific chromosome rearrangements that were present in each population. Chromosome Painting revealed a predominant karyotype for each cell line at the start of the experiment, completed by a large number of variants present in each population. Over time in culture, the predominant karyotype changed for CHO-S and CHO-K1, with the diversity increasing and new variants appearing, while CHO-K1 0 mM Gln preferred chromosome pattern increased in percent of the population over time. As control, Chinese hamster lung fibroblasts were shown to also contain an increasing number of variants over time in culture.

Changes in Chromosome Counts and Patterns in CHO Cell Lines upon Generation of Recombinant Cell Lines and Subcloning.

Chinese hamster ovary (CHO) cells are the number one production system for therapeutic proteins. A pre-requirement for their use in industrial production of biopharmaceuticals is to be clonal, thus originating from a single cell in order to be phenotypically and genomically identical. In the present study it was evaluated whether standard procedures, such as the generation of a recombinant cell line in combination with selection for a specific and stable phenotype (expression of the recombinant product) or subcloning have any impact on karyotype stability or homogeneity in CHO cells. Analyses used were the distribution of chromosome counts per cell as well as chromosome painting to identify specific karyotype patterns within a population. Results indicate that subclones both of the host and the recombinant cell line are of comparable heterogeneity and (in)stability as the original pool. In contrast, the rigorous selection for a stably expressing phenotype generated cell lines with fewer variation and more stable karyotypes, both at the level of the sorted pool and derivative subclones. We conclude that the process of subcloning itself does not contribute to an improved karyotypic homogeneity of a population, while the selection for a specific cell property inherently can provide evolutionary pressure that may lead to improved chromosomal stability as well as to a more homogenous population.

Overexpression of YY1 increases the protein production in mammalian cells.

The production of therapeutic antibodies using mammalian cells remains a high-priority in the biopharmaceutical manufacturing industry. Bioengineers have targeted different cellular processes, including transcription, translation, secretion and post-translational modifications, to overcome the metabolic bottlenecks limiting production capacity and create high-producing mammalian cell lines. The polycomb group (PcG) proteins belong to a family of chromatin regulators with important roles in multicellular development. By overexpressing and screening genes from the PcG family, we have identified an epigenetic key player for biopharmaceutical manufacturing enhancement: the transcription factor Yin Yang 1 (YY1). The overexpression of YY1 led to an increase in the production of several product genes (SEAP, VEGF165, IgG including Rituximab), provided that human YY1 (hYY1) was expressed in human cells (HeLa, HT-1080, HEK-293T, FreeStyle™ 293-F) and Chinese hamster ovary cell-derived YY1 (cYY1) was expressed in CHO cells (CHO-K1, CHO-easyC, FreeStyle™ CHO-S, CHO-B13-24, CHO-IgG1). Ectopic expression of cYY1 in the stable CHO-derived IgG producer cell lines CHO-B13-24 and CHO-IgG1 increased the antibody titer up to 6-fold, suggesting that epigenetic engineering of mammalian production cell lines could become a new strategy to improve the manufacturing of complex protein pharmaceuticals.

The SCF-FBXW5 E3-ubiquitin ligase is regulated by PLK4 and targets HsSAS-6 to control centrosome duplication.

Deregulated centrosome duplication can result in genetic instability and contribute to tumorigenesis. Here, we show that centrosome duplication is regulated by the activity of an E3-ubiquitin ligase that employs the F-box protein FBXW5 (ref. 3) as its targeting subunit. Depletion of endogenous FBXW5 or overexpression of an F-box-deleted mutant version results in centrosome overduplication and formation of multipolar spindles. We identify the centriolar protein HsSAS-6 (refs 4,5) as a critical substrate of the SCF-FBXW5 complex. FBXW5 binds HsSAS-6 and promotes its ubiquitylation in vivo. The activity of SCF-FBXW5 is in turn negatively regulated by Polo-like kinase 4 (PLK4), which phosphorylates FBXW5 at Ser 151 to suppress its ability to ubiquitylate HsSAS-6. FBXW5 is a cell-cycle-regulated protein with expression levels peaking at the G1/S transition. We show that FBXW5 levels are controlled by the anaphase-promoting (APC/C) complex, which targets FBXW5 for degradation during mitosis and G1, thereby helping to reset the centrosome duplication machinery. In summary, we show that a cell-cycle-regulated SCF complex is regulated by the kinase PLK4, and that this in turn restricts centrosome re-duplication through degradation of the centriolar protein HsSAS-6.