Cultured mammalian cells [e.g., murine hybridomas, Chinese hamster ovary (CHO) cells] used to produce therapeutic and diagnostic proteins often exhibit increased specific productivity under osmotic stress. This increase in specific productivity is accompanied by a number of physiological changes, including cell size variation. Investigating the cell size variation of hyperosmotically stressed cultures may reveal, in part, the basis for increased specific productivity as well as an understanding of some of the cellular defense responses that occur under hyperosmotic conditions. The regulation of cell volume is a critical function maintained in animal cells. Although these cells are highly permeable to water, they are significantly less permeable to ionic solutes. Appropriate cellwater content is actively maintained in these cells by regulation of ion and osmolyte balances. Transport appropriate to extracellular conditions, leading to accrual or release of these species, is activated in response to acute cell volume changes. Osmotically induced regulatory volume increases (RVI) and regulatory volume decreases (RVD) are known to occur under a variety of conditions. We observed the time evolution of size variation in populations of two CHO cell lines under hyperosmotic conditions. Observations were made using multiple instruments, multiple cell lines, and multiple cell culture conditions. Size variation of CHO A1 was gauged by flow cytometry using an LSRII 1 flow cytometer while CHO B0 cells were quantified using a Cedex 1 cell analyzer. Hyperosmotic stress had a dose-dependent effect on the regulatory control of cell volume. Stressed cultures of CHO cells grown in suspension exhibited a shift in mean cell diameter. This shift in mean was not due to a change in the whole population, but rather to the emergence of distinct subpopulations of cells with larger cell diameters than those in the bulk of the population. ' MONOCLONAL antibodies (mAbs) play an important role in the modern medical, biological and biotechnological landscape. Not only do they occupy a large and growing segment of new biopharmaceuticals but also they are a lab workhorse providing much of the power for in vitro diagnostics and other essential lab techniques. As demand for these molecules continues to grow, our ability to produce them effectively and efficiently becomes critically important.Many researchers and companies are exploring approaches for increasing production and more broadly reducing costs, which for therapeutic mAbs, can range from 300-3,000 US$ per gram (1). These increases have typically been achieved by optimization of cell culture conditions or optimization of the cell lines themselves. Success in these regimes is determined by increased cell density, increasing cell longevity, and by increasing specific productivity.

The rapidly expanding market for monoclonal antibody and Fc-fusion-protein therapeutics has increased interest in improving the productivity of mammalian cell lines, both to alleviate capacity limitations and control the cost of goods. In this study, we evaluated the responses of an industrial CHO cell line producing an Fc-fusion-protein to hyperosmotic stress, a well-known productivity enhancer, and compared them with our previous studies of murine hybridomas (Shen and Sharfstein, Biotechnol Bioeng. 2006;93:132-145). In batch culture studies, cells showed substantially increased specific productivity in response to increased osmolarity as well as significant metabolic changes. However, the final titer showed no substantial increase due to the decrease in viable cell density. In fed batch cultures, hyperosmolarity slightly repressed the cellular growth rate, but no significant change in productivity or final titer was detected. To understand the transcriptional responses to increased osmolarity and relate changes in gene expression to increased productivity and repressed growth, proprietary CHO microarrays were used to monitor the transcription profile changes in response to osmotic stress. A set of osmotically regulated genes was generated and classified by extracting their annotations and functionalities from online databases. The gene list was compared with results previously obtained from similar studies of murine-hybridoma cells. The overall transcriptomic responses of the two cell lines were rather different, although many functional groups were commonly perturbed between them. Building on this study, we anticipate that further analysis will establish connections between productivity and the expression of specific gene(s), thus allowing rational engineering of mammalian cells for higher recombinant-protein productivity.

Hyperosmotic stress has been shown to increase specific antibody productivity in murine hybridoma systems; however, the mechanisms underlying this phenomenon are still poorly understood. To elucidate the mechanisms for this phenomenon as well as other physiological changes that occur in response to hyperosmotic stress, we performed a genome-wide analysis of the transcriptional response of murine hybridoma OKT3 toward hyperosmotic stress using DNA microarrays. GeneChip MOE430A from Affymetrix was used to determine the differences in transcription patterns between OKT3 in hyperosmotic culture (approximately 100 mOsm above control) and control culture. The chip contains 22,690 probe sets for over 14,000 known genes and more than 4,000 ESTs. Signals were normalized using the GC-RMA algorithm and the effectiveness of hyperosmotic stress in altering the expression of each gene was evaluated using one-way ANOVA. 2,793 probe sets on the chip were differentially expressed with a P < 0.05. Among them, 349 probe sets exhibited a two-fold or greater change (with 202 up-regulated and 147 down-regulated) at one or more time points. Within the 215 characterized, differentially expressed genes, many are involved in metabolism/catabolism (19 induced, 12 repressed), cell-cycle regulation (10 induced, 5 repressed) and apoptosis (8 induced, 2 repressed), regulation of transcription (18 induced, 13 repressed) and translation (2 induced, 2 repressed), transport and signaling pathways (24 induced, 12 repressed). Surprisingly, there were very few changes within the stress-response genes. Interestingly, the transcription levels of both the immunoglobulin kappa and lambda light chains showed a significant change in response to hyperosmotic stress, although there is no detectable lambda chain in the immunoglobulin produced in this cell line. Quantitative PCR assays with TaqMan probes were applied to selected genes to validate the results obtained from microarray analysis.

Abstract:The cellular responses of cultured mammalian cells and non-mammalian organisms to changes in osmolarity are discussed. A number of common themes including activation of protein kinase cascades can be observed in a diverse group of organisms. A combination of physiological and transcriptional studies has been performed to identify regulatory factors and proteins that play a causal role in the cellular responses to osmotic changes. These factors may serve as targets for cellular engineering strategies to improve the productivity of cultured mammalian cells, particularly in response to osmotic shock