• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.293-296
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• 2002

Hyperosmotic pressure increased specific antibody productivity ($q_{Ab}$) of recombinant CHO cells (SH2-0.32) while it depressed cell growth. Thus, the use of hyperosmolar medium did not increase the maximum antibody concentration substantially. To overcome this drawback, the feasibility of biphasic culture strategy was investigated. In the biphasic culture, cells were first cultivated in the standard medium with physiological osmolality(294 mOsm/kg) for cell growth. When cells reached the late exponential phase of growth, the spent standard medium was replaced with the fresh hyperosmolar medium (522 mOsm/kg) for antibody production. The ($q_{Ab}$) in growth phase with the standard medium was 2.1 ${\mu}g/10^6cell/day$ while the ($q_{Ab}$) in antibody production phase with the hyperosmolar medium (522 mOsm/kg) was 11.1 ${\mu}g/10^6cell/day$. Northern blot analysis showed a positive relationship between the relative contenet of Ig mRNA and ($q_{Ab}$), indicating that transcriptional regulation was involved in the response of rCHO cells to hyperosmotic pressure. Due to the enhanced ($q_{Ab}$) and increased cell concentration in biphasic culture, the maximum antibody concentration obtained in biphasic culture with 522 mOsm/kg medium exchange was 161% higher than that obtained in batch culture with the standard medium. Taken together, simple biphasic culture strategy based on hyperosmotic culture for improved foreign protein production from rCHO cells is effective in improving antibody production of rCHO cells.

• KSBB Journal
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• v.31 no.1
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• pp.66-72
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• 2016

Chinese hamster ovary (CHO) cells have been widely used for production of various recombinant proteins such as cytokines and monoclonal antibodies. The cell aggregation and cell death in CHO cell culture directly affect cell viability, and productivity and quality of products. In this study, we investigated preventing effects of storage-protein 2 (SP2) derived from silkworm hemolymph on cell aggregation and cell death in CHO cell culture producing albuminerythropoietin (Alb-EPO). The viable cell density in the culture supplemented with 2 mg/mL SP2 was 1.71-fold higher than that in control culture. Increased titer of Alb-EPO was also found in the culture with SP2. Morphology of CHO cells in SP2 supplemented cultures did not differ from that of control. In addition, the cell aggregation rate of the SP2 cultures was reduced 20% compared to the control. Finally, we confirmed that the apoptosis was strongly suppressed by addition of SP2 in the cultures. These results clearly demonstrate that SP2 can be served as an effective supplement for enhancing titer of Alb-EPO via reducing cell aggregation and cell death.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.257-260
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• 2002

Dissolved oxygen level of cell culture media has a critical effect on cellular metabolism, which governs specific productivity of recombinant proteins and mammalian cell growth However, in the cores of cell aggregates or cell-immobilized beads, oxygen level frequently goes below a critical level. Mammalian cells have a number of genes induced in the lower level of oxygen, and the genes contain a common cis-acting element (-RCGTG-), hypoxia response element (HRE). By binding of hypoxia inducible factor-l (HIF-I) to the HRE, promoters of hypoxia inducible genes are activated, which is a survival mechanism. In this work, to develop a CHO cell capable of producing recombinant proteins in immobilization and high density cell culture efficiently, mammalian expression vectors containing human tissue-type plasminogen activator (t-PA) gene controlled by HRE were constructed and stably transfected into the CHO cells. In $Ba^{2+}$ -alginate immobilization culture, CHO/pCl/dhfr/2HRE-t-PA cells produced 2 folds higher recombinant t-PA activity than CHO/pCl/dhfrlt-PA cells without $CoCl_2$ treatment. Furthermore, in repeated fed batch culture, productivity of t-PA in immobilized CHO/pCI/dhfr/2HRE-t-PA cells was 121 ng/ml/day, total production of 0.968 mg/day at 11 days culture while CHO/pCIIdhfrlt-PA cells was 22.8 ng/ml/day. All these results indicate that HRE is very useful for the enhancement of protein productivity in mammalian cell cultures.

• KSBB Journal
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• v.32 no.2
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• pp.90-95
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• 2017

Mammalian cell cultures have been used extensively to produce proteins for therapeutic agent because of their ability to perform post-translational modification including glycosylation. To produce recombinant protein, many factors and parameter are considered such as media composition, host cell type, and culture process. In this study, recombinant human erythropoietin (rhEPO) producing cell line was established by using glutamine synthetase system. To reduce serum concentration in media, we compared direct adaptation with step adaptation. Cell growth was faster in step adaptation. In low-level serum media, there were insufficient glucose for cell growth. Thus, we added glucose in low-level serum media from 2 g/L to 4.5 g/L. Titer of rhEPO was higher than other conditions at 4.5 g/L of glucose. Additionally, N-methyl-D-aspartate (NMDA), 13-cis-retinal, and pluronic F-68 (PF-68) were added to enhance productivity in CHO cell cultures. In conclusion, we applied CHO cell producing rhEPO to low-level of serum in media using step-adaptation. Also, we confirmed positive effect of NMDA, 13-cis-retinal, and PF-68.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.2
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• pp.123-129
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• 2000

A high cell density culture system for the anchorage dependent CHO cells was developed based on the combination of in removal of ammonium ion and microcarrier culture system, and semi-fed-batch feeding of glucose and glutamine was employed to the developed culture system. The glass bead was selected as an optimum microcarrier in terms of cell growth. An ammonium ion selective zeolite, Phillipsite-Gismondine, was packed in a dialysis menium ion. The semi-fed-batch operation was employer to the novel culture system for the high density cell culture, and the results showed the cell growth was improved by 32% and tPA productivity by 250%.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.217-224
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• 2003

Previously, we developed a CHO cell line (CHO-OTC1-Al9) that expresses the first two enzymes in the urea cycle and exhibits a higher ammonia-removing ability and faster growth rate than a vector-controlled CHO cell line (CHO-neo-5). The current study was undertaken to develop a cell line with an ammonia-removing ability higher than the cell line developed previously. To accomplish this, CHO cell lines expressing the first three, first four, or all five enzymes of the urea cycle were constructed using a stable transfection method. Finally, the CHO-AS-16, CHO-AL-19, and CHO-Arg-11 cell lines expressing the first three, first four, and all five enzymes of the urea cycle, respectively, were selected and found to exhibit higher ammonia-removing ability than the CHO-OTC1-Al9 cell line. Among the three selected cell lines, CHO-AL-19 showed the highest ammonia-removing ability and highest cell viability at a higher cell density, with 40% and 15% lower ammonia concentration in the, culture media than that of CHO-neo-5 and CHO-OTC1-A19 cell lines, respectively. CHO-AL-19 also showed 44% and 10% higher cell viability than the CHO-neo-5 and CHO-OTC-Al9 cell lines, at a higher cell density, respectively. The ammonia concentrations in the culture media were expressed as the ammonia concentration/cell, and the CHO-AL-19 cells revealed 45-60% and 20% lower ammonia concentration/cell than the CHO-neo-5 and CHO-OTC1-Al9 cells, respectively.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.457-458
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• 2001

To determine the effect of ERp57 expression on thrombopoiein (TPO) productivity in recombinant Chinese hamster ovary (rCHO) cells, TPO producing rCHO cell line with doxycycline-regulated ERp57 expression \Vas developed. The Erp57 expression level could be regulated by addition of different concentrations of doxycycline to culture medium. The doxycycline concentration of I ${\mu}g/mL$ was high enough to suppress the ERp57 expression. Up to 5 ${\mu}g/mL$ doxycycline concentration used in culture medium, no observable cytotoxic effect of doxycycline was detected during culture. Overexpression of ERp57 、 vas found to increase the specific TPO productivity ($q_{Tpo}$) without growth inhibition, probably due to the chaperone-like activity of ERp57 in CHO cells.

• Journal of Biomedical Engineering Research
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• v.10 no.2
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• pp.195-202
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• 1989

Chinese Hamster Ovary( CHO) cells were cultured on the surface-modified polymers described in the previous study( "Polymer Surfaces for Cell Adhesion. 1. Surface Modification of Polymers and ESCA Analysis, " J. of KOSOMBE, Vol. 10, No. 1, 43-51, 1989). Among the physicochemical treatment methods. the chloric acid treatment was found to be the best method of rendering the polymer surfaces adhesive for CHO cells probably due to the high density of hydroxyl groups on the surface. Among the biological methods, the fibronectin treatment was best for CHO cell-compatibility probably due to specific active sites existed on the tell-binding domains of the fibronectin structure. When we compare the cell-compatibility of the chloric acid - and the fibronectin -treated PET surfaces, the number of cells attached on the surfaces were increased by 460.5 % and 559.0 % and, respectively, after 32 hr CHO cell culture, compared to that of untreated PET.eated PET.

• KSBB Journal
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• v.22 no.2
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• pp.109-113
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• 2007

Suspension culture of recombinant Chinese hamster ovary (CHO) cells producing follicle-stimulating hormone was performed to investigate the effect of glycine betaine on cell growth and FSH production at low culture temperature. At 28$^{\circ}C$, cell growth was suppressed, but cell viability remained high for a longer culture period. When the culture temperature was lowered from 37$^{\circ}C$ to 28$^{\circ}C$, more than 14-fold increase in the maximum FSH titer was achieved. In batch culture at 28$^{\circ}C$, the use of 15 mM glycine betaine (GB) to culture medium resulted in the enhancement of maximum cell density and FSH titer by 11% and 17%, respectively, compared to the culture without GB. In pseudo-perfusion culture at 28$^{\circ}C$ with the exchange of fresh medium containing 15 mM GB, a final FSH of $2,058{\mu}g$ which is approximately 1.4-fold higher as compared to the culture without GB was obtained. This enhanced FSH production with 15 mM GB was not just because of enhanced specific FSH productivity (qFSH), but mainly because of the extended culture longevity. Taken together, this result demonstrates that the application of GB at low culture temperature is feasible to enhance the production of recombinant proteins in rCHO cells.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.66-69
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• 2001

Previously we developed a CHO cell line (CHO-OTC1-A19) expressing the first two enzymes of urea cycle. This cell line showed higher ammonia removal activity and faster growth rate than the vector controlled CHO cells (CHO-neo-5). The purpose of this study was to develop a cell line with higher ammonia removal activity than the cell line developed previously. To accomplish this, we constructed stable CHO cell lines expressing the first three, the first four, or all five enzymes of urea cycle by the stable transfection method. We finally selected CHO-AL-19 cell line expressing the first three, the first four enzymes of the cycle with higher ammonia activity than CHO-OTC1-A19 and CHO-n대-5 cell lines: 40% and 15% higher than those of CHO-neo-5 and CHO-OTC1-A19 cell lines 72 hour after culture started, respectively. It also showed 44% and 10% higher cell viability than CHO-neo-5 and CHO- OTC1-A19 cell lines at higher cell density. In addition, CHO-AL-19 cells showed 45%-60% and about 20% lower ammonia concentration per cell than those of CHO-neo-% and CHO-OTC1-A19 cell lines, respectively. These results indicate that CHO-AL-19 could be used in the production of human therapeutic proteins with higher efficiency.