Manipulation of Heterogeneous Hybridoma Cultures for Overproduction of Monoclonal Antibodies

McKinney, Katherine L.; Dilwith, Robert L.; Belfort, Georges

doi:10.1021/bp00011a010

Cited by 46 publications

(32 citation statements)

References 33 publications

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“…Cell Cycle, Differentiation, and Death. In an effort to improve antibody productivity, many investigators have explored the roles of growth 31,32 on antibody production; the major emphasis was on cell cycle kinetics. [33][34][35] Their results were quite variable.…”

Section: Transcriptional Changes Upon Application Of Hyperosmotic Stressmentioning

confidence: 99%

Transcriptomic responses to sodium chloride‐induced osmotic stress: A study of industrial fed‐batch CHO cell cultures

Shen

Kiehl

Khattak

et al. 2010

Biotechnology Progress

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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.

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Section: Transcriptional Changes Upon Application Of Hyperosmotic Stressmentioning

confidence: 99%

Transcriptomic responses to sodium chloride‐induced osmotic stress: A study of industrial fed‐batch CHO cell cultures

Shen

Kiehl

Khattak

et al. 2010

Biotechnology Progress

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show abstract

“…In an effort to improve antibody productivity, many investigators have explored the roles of growth (Dalili and Ollis, 1990;McKinney et al, 1991) on antibody production; the major emphasis was on cell cycle kinetics (Hayter et al, 1992;Kromenaker and Srienc, 1994;Park and Ryu, 1994). Their results were quite variable.…”

Section: Cell Proliferation and Deathmentioning

confidence: 99%

Genome‐wide analysis of the transcriptional response of murine hybridomas to osmotic shock

Shen¹,

Sharfstein²

2005

Biotech & Bioengineering

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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.

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“…Another study found a correlation between mean surface fluorescence and total cell-associated IgG as determined by ELISA; however, they were unable to find a relationship between surface fluorescence and specific productivity (Meilhoc et al 1989). In contrast, when cells with a higher surface fluorescence were sorted from the population as a whole, they had a higher specific productivity than the entire culture or cells with a lower surface fluorescence (McKinney et al 1991). However, there was no correlation between surface fluorescence and intracellular antibody as measured by fluorescent staining.…”

Section: Introductionmentioning

confidence: 93%

“…Many investigators have explored the roles of growth (Dalili and Ollis 1990;Heath et al 1990;McKinney et al 1991) and, more specifically, cellcycle kinetics (Al-Rubeai and Emery 1990;Hayter et al 1992;Kromenaker and Srienc 1994;Park and Ryu 1994) on antibody production in an effort to improve antibody productivity. The results of these studies were quite variable.…”

Section: Introductionmentioning

confidence: 99%

Techniques for Dual Staining of DNA and Intracellular Immunoglobulins in Murine Hybridoma Cells: Applications to Cell-Cycle Analysis of Hyperosmotic Cultures

2005

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Flow cytometry was used to evaluate the effects of hyperosmotic stress on cell-cycle distribution and cell-associated immunoglobulins for murine hybridoma cells grown in batch culture. Paraformaldehyde/methanol fixation substantially increased the fluorescence signal for intracellular immunoglobulins compared to ethanol fixation. For surface immunoglobulins, similar fluorescence signals were observed regardless of fixation method. Dual staining of immunoglobulins and cellular DNA was employed to determine immunoglobulin pool size as a function of cell-cycle phase. The intracellular immunoglobulin pool sizes increased as the cells progressed through the cell cycle for both control and hyperosmotic cultures. For control cultures, the immunoglobulin pool size increased during the exponential phase of culture, followed by a decrease as the cultures entered stationary phase. In contrast, hyperosmotic cultures showed an initial decrease in immunoglobulin pool size upon the application of osmotic shock, followed by an increase to a level above that of control cultures. This behavior was observed in all phases of the cell cycle. In addition, hyperosmotic cultures exhibited an increase in cell size when compared to control cultures. When normalized for cell size, the intracellular immunoglobulin concentration in hyperosmotic cultures was initially lower than in control cultures and subsequently increased to slightly above the level of control cells. Cells in all phases of the cell cycle behaved in a similar manner. There was no apparent relationship between the intracellular antibody concentration and the rate of antibody secretion.

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Manipulation of Heterogeneous Hybridoma Cultures for Overproduction of Monoclonal Antibodies

Cited by 46 publications

References 33 publications

Transcriptomic responses to sodium chloride‐induced osmotic stress: A study of industrial fed‐batch CHO cell cultures

Transcriptomic responses to sodium chloride‐induced osmotic stress: A study of industrial fed‐batch CHO cell cultures

Genome‐wide analysis of the transcriptional response of murine hybridomas to osmotic shock

Techniques for Dual Staining of DNA and Intracellular Immunoglobulins in Murine Hybridoma Cells: Applications to Cell-Cycle Analysis of Hyperosmotic Cultures

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