Recent technological advances in cell line and bioprocess development have driven significant improvements in product titers and enabled scientists to accelerate product development timelines (1). Despite those successes, many limitations in developing cell lines for biotherapeutics remain. One example in fed-batch cultures is an apparent paradox: when cell growth is inhibited by high osmolarity after multiple additions of concentrated nutrients intended to enhance cell growth and protein production. Generation of novel host cells to overcome specific bottlenecks found in bioprocessing is highly desirable.

Imposing hyperosmotic stress conditions on commercially popular production lines such as Chinese hamster ovary (CHO) and NS0 has previously been shown to improve specific productivity (2,3). That appears to correlate with cellular changes in transcription, translation, protein secretion, and metabolism (4,5). Shen and Sharfstein analyzed transcriptional response to osmotic shock using DNA microarray and RT-PCR techniques (6), and Yee et al. applied DNA microarray and two-dimensional gel electrophoresis to analyze proteomic profiles in response to sodium butyrate treatment (7). Although several investigators have reported that specific productivity can be enhanced by higher osmolarity, the overall protein yield was not substantially improved because of poor cell viability and growth when osmolarity exceeded 420–450 mOsm/kg. Kim and Wu, et al. speculated the cause to be cell growth inhibition and apoptosis induction from osmolarity pressure (8,9). Although these studies have contributed to elucidating the effect of a high-osmolarity environment on cell behavior and cellular processes, few investigators have explored whether altering a host cell can help it overcome a high-osmolarity environment.

PRODUCT FOCUS: All Biologics

PROCESS FOCUS: Production

WHO SHOULD READ: Product and process development

KEYWORDS: CHO DG44, fed-batch culture, osmolarity resistance, nutrient feeding strategy, cell line engineering

LEVEL: INTRODUCTION

Adapting host cells to overcome other environmental or bioprocessing obstacles has been explored for limited and specific applications. For example, adapting them to defined media formulations is widely reported, with recent emphasis on novel media components and animal-origin free raw materials (10,11). Berdichevsky et al. successfully isolated a faster-growing cell population after extensive passaging and adaptation to improve virus production (12). Timelines for such spontaneous adaptation events tend to be lengthy, however, and efforts may be unsuccessful especially for isolating rare biological traits.

Genetic engineering is a more direct approach for host-cell improvement. Successful examples include cell growth enhancement through introduction of cell cycle control, growth factor, and anti-apoptosis genes (13). Other host improvements have improved protein quality by overexpressing glycosyltransferase/glycosidase genes (14) or knocking out the fucosyl transferase gene (15). These approaches often require extensive knowledge of the genes and cellular mechanisms involved.

To overcome the various technical constraints, we investigated whether novel host cells could be generated by a new methodology that first produces a genetically diverse cell population from which cells exhibiting a rare or difficult-to-find phenotype then can be screened and isolated. Generation of the genetically diverse population is achieved by temporarily suspending the DNA mismatch repair (MMR) mechanism of dividing cells (16). Screening and isolation are achieved by applying environmental selective pressure.

The approach of disrupting MMR is based on introducing the truncated hPMS2-134 gene: the Revolution gene (Figure 1), which is an allelic variant of the human PMS2 gene product (17,18). CHO cells harboring this genetic variant have been shown to accumulate mutations at significantly higher frequencies (100–500 fold enhancement) than controls (16). The nontoxic nature of this approach also resulted in robust cells that could progress through cell cycle checkpoints with virtually no loss in viability (16,19). The outcome is generation of a hardy and broad library of cells from which subclones with desired characteristics can be screened and isolated (20,21). After such isolation, the gene can be removed using a negative selection marker for restoration of the MMR mechanism (16).

To test our approach, we used CHO DG44 as our model cell line to evaluate whether genetic diversity can be introduced through Revolution transfection and whether a novel cell population resistant to high osmolarity could be isolated. We also tested whether this isolated phenotype was robust and stable and whether the host cells could be transfected to express a monoclonal antibody under fed-batch conditions for future bioprocessing applications.