• Biotechnology and Bioprocess Engineering:BBE
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• 제6권5호
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• pp.332-336
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• 2001

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 (q$\_$Tpo/)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 in q$\_$Tpo/ 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 enhanced q$\_$Tpo/. When the enhancement of the q$\_$Tpo/ of 9 clones after a high MTX dosage was plotted against the extent of the increase of their size, there was a linear correlation (γ$^2$=0.80, p<0.001, ANOVA), which suggested that an enhancement of q$\_$Tpo/ 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 enhanced q$\_$Tpo/ can be done upon their increased size and growth pattern. This facilitates the development of highly productive recombinant CHO cell lines.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVI)
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• pp.210-213
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• 2005

본 연구에서는 세포 사멸을 억제시켜 재조합 단백질의 생산을 향상시키기 위한 전략을 개발하기 위해, 우선 STR-G와 EGCG를 농도별로 첨가하여 세포 성장, 사멸 그리고 IDS 생산성에 미치는 영향을 조사하였다. Flow cytometry 분석 결과, STR-G에 의한 apoptotic cell percentage의 감소, STR-G와 EGCG에 의한 bel-2 expression level의 증가와 IDS 생산성의 증가를 확인하였다.

• KSBB Journal
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• 제32권3호
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• pp.193-198
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• 2017

Sialylation is important in producing therapeutic proteins such as antibody, cytokine and fusion protein. Thus, enhancement of sialylation is usually performed in CHO cell cultures. ${\alpha}2,6$-Sialyltransferase (ST), which plays a key role in the attachment of ${\alpha}2,6-sialic$ acid, is present in human cells but not in Chinese hamster ovary (CHO) cells. Overexpression of ${\alpha}2,6-ST$ can be used for enhancing the degree of sialylation and achieving human-like glycosylation. In this study, we constructed CHO cells producing human cytotoxic T-lymphocyte antigen4-immunoglobulin (hCTLA4-Ig) as well as ${\alpha}2,6-ST$. Transfected CHO cells were selected using G418 and stable cell line was established. Profiles of viable cell density and hCTLA4-Ig titer in an overexpressed cell line were similar to those of a wild-type cell line. It was confirmed that the total amount of sialic acid was increased and ${\alpha}2,6-sialic$ acid was attached to the terminal residues of N-glycan of hCTLA4-Ig by ESI-LC-MS. Compared to 100% of ${\alpha}2,3-sialic$ acid in wild type cells, 70.9% of total sialylated N-glycans were composed of ${\alpha}2,6-sialic$ acid in transfected cells. In conclusion, overexpression of ${\alpha}2,6-ST$ in CHO cells led to the increase of both the amount of total sialylated N-glycan and the content of ${\alpha}2,6-sialic$ acid, which is more resemble to human-like structure of glycosylation.

• Journal of Microbiology and Biotechnology
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• 제23권8호
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• pp.1176-1184
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• 2013

Anti-hepatocyte growth factor (anti-HGF) monoclonal antibodies (mAbs) are potential therapeutics against various cancers. Screening for high-producer clones is a time-consuming and complex process and is a major hurdle in the development of therapeutic mAbs. Here, we describe an efficient approach that allows the selection of high-producer Chinese hamster ovary (CHO) cell lines producing the novel anti-HGF mAb SFN68, which was generated previously by immunizing HGF bound to its receptor c-Met. We selected an SFN68-producing parental cell line via transfection of the dihydrofolate reductase-deficient CHO cell line DG44, which was preadapted to serum-free suspension culture, with an SFN68-expression vector. Subsequent gene amplification via multiple passages of the parental cell line in a methotrexate-containing medium over 4 weeks, followed by clonal isolation, enabled us to isolate two cell lines, 2F7 and 2H4, with 3-fold higher specific productivity. We also screened 72 different media formulated with diverse feed and basal media to develop a suboptimized medium. In the established suboptimized medium, the highest anti-HGF mAb yields of the 2F7 and 2H4 clones were 842 and 861 mg/l, respectively, which were about 10.5-fold higher than that of the parental cell line in a non-optimized basal medium. The selected CHO cell lines secreting high titers of SFN68 would be useful for the production of sufficient amounts of antibodies for efficacy evaluation in preclinical and early clinical studies.

• Biotechnology and Bioprocess Engineering:BBE
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• 제6권2호
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• pp.117-127
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• 2001

The high-level expression of a human single chain urokinase-type plasminogen activator (scu-PA) was achieved by employing a methotrexate (MTX)-dependent gene amplification system in Chinese hamster ovary (CHO) cells. By cotransfecting and coamplifying a scu-PA expression plasmid and dihydrofolate reductase (DHFR) minigene, several scu-PA expressing CHO cell lines were selected and gene-amplified. These recombinant cell lines, NGpUKs, secreted a completely processed scu-PA of 54 kD and up to 60mg/L was accumulated in the culture medium when they were adapted to an optimal MTX concentration. Over 95% of the scu-PA expressed was secreted in the culture medium and identified having the proper function of a plasminogen activator when activated by plasmin. Based on a genomic Southern analysis, a representative subclone, MGpUK-5, exhibited MTX-dependent scu-PA gene amplification, plus the initial single-copy gene of scu-PA eventually turned into about 150 copies of the amplified gene of scu-PA after gradual adaptation to 2.0$\mu$M of MTX. Meanwhile, the transcripts kof the scu-PA gene increased, although -early saturation of transcription was identified at 0.1$\mu$M of MTX. The scu-PA production by the MGpUK-5 subclone also increased relative to the gene amplification and increased transcripts, however, the relationship was not linearly proportional. Accordingly, since the MGpUK cell lines expressed elevated levels of enzymatically active scu-PA, these cell lines could be applied to the largescale production of scu-PA.

• 한국미생물·생명공학회지
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• 제16권4호
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• pp.282-286
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• 1988

Chinese Hamster Ovary 세포의 대량배양을 위한 동력학적 변수들을 산소 소비속도의 on-line 측정으로 구하였다. 세포성장과 산소 소비속도의 상관관계 계수가 0.83으로 밀접한 상호 직선관계가 있음을 입증하며, 이에 근거를 둔 model에 의해 간접 계산된 세포수가 실제 측정된 세포수와 상당히 일치하였다. 따라서 세포끼리 서로 뭉쳐 직접 세포수 측정이 부정확한 경우 산소 소비속도의 측정에 의해 간접적으로 세포수를 예측할 수 있음을 입증하였다. 산소 세포수율, Yx/o와 mass transfer coefficient, $K_{\ell}$a가 각각 1.26$\times$$10^4$cells/mmole $O_2$ consumed와 1.011/h로 측정되었으며, 평균 세포 성장속도는 2.891/day로 계산되었다. 이는 매일 2 gram의 tPA를 연속 생산할 수 있는 조건이었다.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.712-720
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• 2007

To investigate clonal variations of recombinant Chinese hamster ovary(rCHO) clones in response to culture pH and temperature, serum-free suspension cultures of two antibody-producing CHO clones(clones A and B), which were isolated from the same parental clone by the limiting dilution method, were performed in a bioreactor at pH values in the range of 6.8-7.6, and two different temperatures, $33^{\circ}C\;and\;37^{\circ}C$. In regard to cell growth, clone A and clone B displayed similar responses to temperature, although their degree of response differed. In contrast, clones A and B displayed different responses to temperature in regard to antibody production. In the case of clone A, no significant increase in maximum antibody concentration was achieved by lowering the culture temperature. The maximum antibody concentration obtained at $33^{\circ}C$(pH 7.4) and $37^{\circ}C$(pH 7.0) were $82.0{\pm}2.6$ and $73.2{\pm}4.1{\mu}g/ml$, respectively. On the other hand, in the case of clone B, an approximately 2.5-fold increase in maximum antibody concentration was achieved by lowering the culture temperature. The enhanced maximum antibody concentration of clone B at $33^{\circ}C$($132.6{\pm}14.9{\mu}g/ml$ at pH 7.2) was due to not only enhanced specific antibody productivity but also to prolonged culture longevity. At $33^{\circ}C$, the culture longevity of clone A also improved, but not as much as that of clone B. Taken together, CHO clones derived from the same parental clone displayed quite different responses to culture temperature and pH with regards antibody production, suggesting that environmental parameters such as temperature and pH should be optimized for each CHO clone.

• 대한의용생체공학회:의공학회지
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• 제10권2호
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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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• 제31권4호
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• pp.270-276
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• 2016

In glycoprotein, Terminal sialic acid residues of N-linked glycan are imperative things because they prevent the recognition from asialoglycoprotein-receptor that affect the half-life of glycoproteins. So establishment of culture process for enhancing sialic acid is important to maximize sialic acid contents of glycoprotein. In this study, we investigated effects of biphasic culture of Chinese hamster ovary (CHO) cells producing albumin-erythropoietin to increase sialylation. Biphasic cultures were performed with shift of $CO_2$ concentrations and temperatures at day 5 when viable cell density was decreased and sialidase was started to be released by cell lysis. The examined temperature set points were 33, 35 and $37^{\circ}C$ respectively and the $CO_2$ concentration was 1, 5, 10 and 15%. We confirmed that sialidase activity was the lowest in biphasic culture that was shifted from normal culture condition to 1% of $CO_2$ and $33^{\circ}C$ on day 5. However, the temperature and concentration of $CO_2$ have little effect on activity of ${\alpha}2,3$-sialyltransferase. Also, sialic acid contents were enhanced 1.13-fold higher than that in control culture. In conclusion, Biphasic cultivation in CHO cells led to inhibition of sialidase activity and increases of sialylated glycan.

• Applied Biological Chemistry
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• 제50권2호
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• pp.132-135
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• 2007

국내에서 새로 육성된 장미의 부가가치를 향상시키고 천연 식품소재로서 기능성을 탐색하는 기초연구의 일환으로 5개 장미 품종을 대상으로 유기용매 추출물을 제조하고 장미꽃 추출물이 CHO 세포의 생육에 미치는 영향을 MTT법으로 조사하였다. 장미 추출물들은 추출용매의 종류와 첨가농도에 따라 CHO 세포의 증식을 촉진하거나 생육을 억제하였으며 품종에 따른 차이는 없었다. Ether로 추출한 장미꽃 추출물을 5 ${\mu}$g/ml 농도로 첨가한 경우 대조군에 비하여 CHO 세포의 증식촉진효과가 가장 높았다. 이러한 결과는 장미꽃은 CHO 세포의 증식을 조절하는 생리활성을 나타내며, 신 기능성 식품소재로서 활용하는 가능성을 시사한다.