Influence of genetic factors on the productivity of recombinant Chinese hamster ovary (CHO) cells

Citation

Pendse, Girish J. (1992) Influence of genetic factors on the productivity of recombinant Chinese hamster ovary (CHO) cells. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/60ap-a030. https://resolver.caltech.edu/CaltechETD:etd-08132007-103800

Abstract

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In this work, influence of some genetic factors on the productivity of recombinant Chinese hamster ovary (CHO) cells has been investigated using a number of experimental techniques. The first system that we have studied is a CHO cell line producing Hepatitis B surface antigen (HbsAg). We have determined the effect of cloned gene dosage on cell growth and HbsAg synthesis and secretion. Gene copy numbers in each clone determined using Southern hybridizations were positively correlated with intracellular dihydrofolate reductase (DHFR) content using a flow cytometric assay. The mRNA levels quantitated using Northern hybridization followed by autoradiography and densitometry also gave the same trends. Flow cytometry experiments also show that the amplified clones exhibit a great deal of heterogeneity in single-cell DHFR content as compared to parental cells which were quite homogeneous with respect to intracellular DHFR content.

Batch culture experiments carried out in T flasks show that specific growth rates decrease with gene copy number. Secreted HbsAg titers and specific HbsAg secretion rates were found to increase with gene copy number. Specific glucose and glutamine consumption rates as well as specific lactate and NH3 production rates did not vary significantly between the clones although the 1 [...] clone which has the highest relative gene copy number among the clones we studied, exhibited metabolic rates that were slightly higher than those for the other clones.

Using pulse-chase experiments we found that efficiency of HbsAg secretion (fraction of total initial HbsAg (0 h chase) that is secreted into the extracellular medium during a 23.5 h chase) decreases and overall efficiency of HbsAg expression (relative amount of HbsAg secreted into the medium, [AU.mm/[...] cells]/relative gene copy number) decreases whereas intracellular HbsAg degradation increases with cloned gene dosage. Comparisons between the relative gene copy number, relative mRNA level, relative amount of total initial HbsAg (0 h chase) and HbsAg secreted into the medium during the longest chase time of 23.5 h for various clones indicate that neither transcription nor translation is limiting at high cloned gene dosage. Our results show that some step after translation limits the overall productivity of these cells at high cloned gene dosage.

[...] NMR spectroscopy was carried out using a novel continuous flow packed bed reactor configuration to study the intracellular metabolism of recombinant CHO cells. NMR measurements of CHO cells growing in macroporous collagen microspheres indicate that intracellular phosphorylated metabolite levels including NTP is higher in cells producing HbsAg. These transformed cells producing HbsAg, which exhibit lower specific growth rate in the packed bed, may be utilizing these higher NTP amounts in the HbsAg secretory pathway instead of the cellular biosynthetic pathway.

The second system studied here is a CHO cell line producing secreted tissue plasminogen activator (tPA). ATP limitations may influence productivity of secreted cloned proteins in CHO cells and expression of Vitreoscilla hemoglobin (VHb) has been suggested to increase ATP production efficiency in Escherichia coli. Based on these hypotheses, we have cloned the Vitreoscilla hemoglobin gene in CHO cells producing tPA (CHO-tPA) and obtained inducible intracellular expression of VHb in these transfected cells (VHb-CHO). We confirmed the presence of the VHb gene in the genomic DNA of these cells by Southern blots while Western blots were used to demonstrate the expression of VHb protein upon induction with dexamethasone. Batch culture experiments were carried out in order to determine the effect of VHb expression on growth and tPA secretion. We have shown that VHb-CHO cells have a lower specific growth rate compared to CHO-tPA cells but exhibit a significantly higher specific tPA production rate throughout the batch.

We have also investigated the effect of some growth factors on cell growth and monoclonal antibody productivity in batch hybridoma cultures. Growth factor addition did not affect specific growth rate of the cells significantly, but significant variation in monoclonal antibody production occurred depending on the growth factor present in the medium. These results indicate that growth factors affect some step in the monoclonal antibody synthesis and secretion pathway without significantly influencing the cell specific growth rate.

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