Recombinant Chinese Hamster Ovary Cell Lines Research Articles

Overexpression of cold‐inducible RNA‐binding protein increases interferon‐γ production in Chinese‐hamster ovary cells

Culturing recombinant CHO (Chinese-hamster ovary) cells at low temperatures (30-33 degrees C) increases specific recombinant protein productivity by 2-5-fold. However, even though the specific productivity is increased, cell growth is decreased in low-temperature culture such that the final recombinant protein titre remains unchanged or is even diminished, owing to the lower cell density. Exposing mammalian cells to low temperatures results in a change in the expression of many 'cold-stress' genes. CIRP (cold-inducible RNA-binding protein) is a cold-stress protein that is highly expressed at 32 degrees C, but not at 37 degrees C. In the present study we demonstrated that overexpression of CIRP at 37 degrees C can increase the recombinant-protein titre in CHO cells. Stable overexpression of CIRP at 37 degrees C improved the final titre of CHO IFN-gamma, a recombinant CHO cell line producing human IFN-gamma (interferon-gamma), by 25% in adherent culture and up to 40% in suspension culture. Real-time PCR analysis showed that the increase in the recombinant IFN-gamma titre could be attributed to increased recombinant IFN-gamma mRNA levels, while growth data showed that CIRP overexpression did not result in growth arrest in CHO IFN-gamma cells. Glycan analysis showed that the increase in IFN-gamma titre as a result of CIRP overexpression did not affect the site occupancy, glycan structures or sialic acid content of IFN-gamma. Using this strategy, the final IFN-gamma titre was increased by 40% compared with current temperature-based strategies. Furthermore, there is no decrease in cell growth or recombinant-protein glycosylation quality, as previously observed in low-temperature culture.

Separation of CHO cells using hydrocyclones

Hydrocyclones are simple and robust separation devices with no moving parts. In the past few years, their use in animal cell separation has been proposed. In this work, the use of different hydrocyclone configurations for Chinese hamster ovary (CHO) cell separation was investigated following an experimental design. It was shown that cell separation efficiencies for cultures of the wild-type CHO.K1 cell line and of a recombinant CHO cell line producing granulocyte-macrophage colony stimulating factor (GM-CSF) were kept above 97%. Low viability losses were observed, as measured by trypan blue exclusion and by determination of intracellular lactate dehydrogenase (LDH) released to the culture medium. Mathematical models were proposed to predict the flow rate, flow ratio and separation efficiency as a function of hydrocyclone geometry and pressure drop. When cells were monitored for any induction of apoptosis upon passage through the hydrocyclones, no increase in apoptotic cell concentration was observed within 48 h of hydrocycloning. Thus, based on the high separation efficiencies, the robustness of the equipment, and the absence of apoptosis induction, hydrocyclones seem to be specially suited for use as cell retention devices in long-term perfusion runs.

Relationship between cell size and specific thrombopoietin productivity in chinese hamster ovary cells during dihydrofolate reductase-mediated gene amplification

When parental Chinese hamster ovary (CHO) cell clones that are capable of producing thrombopoietin (TPO) were subjected to high methotrexate (MTX) concentrations, clonal variations in cell growth were apparent. In the clones that had no significant enhancement in specific TPO productivity (qTpo) when a higher level of MTX was administered, their growth was not depressed significantly nor their cell size changed significantly. On the other hand, those clones that showed a significant enhancement inqTpo at higher a MTX dosage, cell growth was depressed initially but recovered during successive sub-cultures. Furthermore, their cell size increased, which suggested that changes in cell size may be indicative of an enhancedqTpo. When the enhancement of theqTpo of 9 clones after a high MTX dosage was plotted against the extent of the increase of their size, there was a linear correlation (r2=0.80,P<0.001, ANOVA), which suggested that an enhancement ofqTpo after high MTX administration can be measured by the increase in their cell size. Taken together, our data demonstrate that the selection of amplified CHO cell clones with enhancedqTpo can be done based upon their increased size and growth pattern. This facilitates the development of highly productive recombinant CHO cell lines.

Tissue plasminogen activator coexpressed in Chinese hamster ovary cells with alpha(2,6)-sialyltransferase contains NeuAc alpha(2,6)Gal beta(1,4)Glc-N-AcR linkages.

Genetic alteration of the set of oligosaccharide biosynthesis enzymes expressed in a genetically engineered host cell line is a plausible strategy for manipulating the oligosaccharides on a cloned glycoprotein coexpressed in that cell line. This hypothesis was verified for the particular case of sialylation of recombinant human tissue plasminogen activator (tPA) expressed by an engineered Chinese hamster ovary (CHO) cell line. The gene for rat liver beta-galactoside alpha(2,6)-sialyltransferase (2,6-ST) was cloned behind the MMTV promoter in the vector pMSG and transfected into a tPA-expressing CHO cell line. Selected and screened transfectants exhibited significantly greater surface fluorescence than controls in flow cytometric analyses of cells labeled with Sambacus nigra agglutinin (SNA)-biotin and streptavidin-R-phycoerythrin; SNA specifically binds to NeuAc alpha(2,6)Gal beta(1,4)Glc-N-AcR linkages, which are synthesized by 2,6-ST and which are not normally found on CHO cells. SNA blots of partially purified tPA from the culture supernatant demonstrated that tPA synthesized in the 2,6-ST transfectants possessed terminal NeuAc alpha(2,6)Gal beta(1,4)Glc-N-AcR linkages, while tPA from the original recombinant CHO cell line did not. Besides possibly allowing the production of glycoproteins in cell culture with glycosylation more closely resembling that in humans, extensions of this strategy have the potential to tailor the pharmacokinetics, targeting, and antigenic properties of cloned glycoproteins.