Hyperosmotic Stress in Murine Hybridoma Cells: Effects on Antibody Transcription, Translation, Posttranslational Processing, and the Cell Cycle

Abstract

Mechanisms for increased antibody production in batch cultures of murine hybridoma cells in response to hyperosmotic stress were investigated. The rates of immunoglobulin transcription and protein translation and posttranslational processing were determined in control and hyperosmotic cultures. Changes in immunoglobulin transcription played a minor role in the increase in antibody production in response to hyperosmotic stress. In contrast, protein translation increased substantially in response to osmotic stress. However, the antibody translation rate remained relatively constant after correcting for the overall increase in protein translation. Cell size and intracellular antibody pool also increased in response to hyperosmolarity. The intracellular antibody pool increased proportionately with the increase in cell size, indicating that hyperosmotic cultures do not selectively increase their intracellular antibody population. Changes in cell cycle distribution in response to osmotic stress and the relationship between the cell cycle and antibody production were also evaluated. Hyperosmotic stress altered the cell cycle distribution, increasing the fraction of the cells in S-phase. However, this change was uncorrelated with the increase in antibody production rate. Immunoglobulin degradation was relatively low ( approximately 15%) and remained largely unchanged in response to hyperosmotic stress. There was no apparent increase in immunoglobulin stability as a result of osmotic stress. Antibody secretion rates increased approximately 50% in response to osmotic stress, with a commensurate increase in the antibody assembly rate. The rate of transit through the entire posttranslational processing apparatus increased, particularly for immunoglobulin light chains. The levels of endoplasmic reticulum chaperones did not increase as a fraction of the total cellular protein but were increased on a per cell basis as the result of an increase in total cellular protein. A difference in the interactions between the immunoglobulin heavy chains and BiP/GRP78 was observed in response to hyperosmotic conditions. This change in interaction may be correlated with the decrease in transit time through the posttranslational pathways. The increase in the posttranslational processing rate appears to be commensurate with the increase in antibody production in response to hyperosmotic stress.

Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line

“…An additional optimization strategy would be prevention of the onset of apoptosis after cessation of growth, at which time cells are accumulating in the G1 cell cycle phase. Identification of regulatory mechanisms for cell cycle progression and of associated environmental manipulation are emerging areas of research (Balcarcel and Stephanopoulos 2001;Bjorklund et al 2006;Carvalhal et al 2003;deZengotita et al 2001;Ibarra et al 2003;Sun et al 2004;Sunstrom et al 2000).…”

Downregulation of NFAT5 by RNA interference reduces monoclonal antibody productivity of hybridoma cells

“…The transcription levels of both heavy chain (HC) and light chain (LC) mRNAs are enhanced as well as antibody productivity [6]. The increase of the post-translational processing rate appears to be commensurate with that of antibody production in response to hyperosmotic stress [7]. Recently, a genome-wide analysis has been performed to investigate the transcriptional response of hybridoma cells towards hypertonic stress using DNA microarrays [8].…”

Scale-down of CHO cell fed-batch cultures

“…Kim et al 58 reported that the enhancement of the specific thrombopoietin productivity is linearly correlated with the increase in cell size for nine CHO cell clones generated using the same transfection process. In a number of studies the relation between cell size and specific productivity was used to enhance mAb productivity, by directly or indirectly increasing the cell size, for example, through cell cycle arrest 53,91 , hyperosmotic pressure 92,93 , and mild hypothermia 57,94 . Acetic acid and formic acid accumulation were observed during the cultures (Figure 2b).…”

“…The result agrees with the fact that after day 5 cell proliferation has stopped in the ActiCHO process (Figure 1). In several CHO cell culture processes, hypothermia 19,127,128 , hyperosmotic stress 92,129 , and cell cycle inhibitors 53,91,130 were applied that mediated a G0/G1 arrest without influencing G2/M phase. This was, however, not the case in the present study where cell cycle arrest occurred in both G0/G1 and the G2/M phase.…”

“…To obtain an increased volumetric productivity without increasing cell density, an increase in specific productivity (qp) is needed. For this, several strategies have been described, including hyperosmotic pressure 92 , cell cycle arrest 53 , and mild hypothermia 57 . In these studies, the increase in qp was found to be correlated with an increase in cell size.…”