• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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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.

• KSBB Journal
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• v.9 no.1
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• pp.48-54
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• 1994

A modified amidolytic assay and a fibrin plate method were used to accurately measure the concentration of single chain urokinase type plasminogen activator (scu-PA) and two-chain urokinase type plasminogen activator (tc-PA) in the spent media. $1.65{\times}10^6$(viable cells/ml) of maximum cell density and 1670(IU/ml) of scu-PA concentration were obtained in 1% serum containing medium. The overall conversion ratio from scu-PA to tc-PA was less than 10%. In the results of batch cultivation in a spinner vessel, $4.43{\times}10^6(total cells/ml)$ of maximum cell density and 1560(IU/ml) of scu-PA concentration was observed. The maximum scu-PA concentration and specific scu-PA Productivity were obtained in 1760(IU/ml) and $3.13{\times}10^{-4}(IU/cell)$, respectively, from perfusion cultivation. The conveysion ratios from batch, fed-batch and perfusion cultivations were less than 12%, which means that about 90% of scu-PA secreted from the cells can be maintained during the cultivations.

• KSBB Journal
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• v.9 no.5
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• pp.518-524
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• 1994

Serum free medium was developed to cultivate Human Embryonic Kidney cells with Cytodex III microcarriers. The cells normally attached and spreaded on the microcarriers in serum free medium, and grew well by bead to bead processes. 85% of attachment yield was obtained on Cytodex III in this medium, compared to about 93% in 1% serum containing medium. About 90% of the attached HEK cells spreaded after 6 hours of post-attachment periods on the surface of microcarrier. Maximum cell density and scu-PA concentration in a serum free medium were $9.1{\times}10^5$ cells/ml and 1790 IU/ml, respectively, with fed-batch cultivation. Maximum cell density and scu-PA concentration in this medium with perfusion cultivation were $2.5{\times}10^6$ cells/ml and 1820 IU/ml, respectively. The conversion of single chain urokinase type plasminogen activator (scu-PA) into two chain type plasminogen activator (tc-PA) was less than 5% in a serum free medium compared to about 10% in 0.5% serum containing medium.

• KSBB Journal
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• v.5 no.4
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• pp.391-396
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• 1990

4.0$\times$10-3 (IU/cells/day) of maximum specific scu-PA production, which was higher than those by a 75$\textrm{cm}^2$ T-flask and conventional perfusion systems, was maintained by tube-type bioreactor at 0.35 (1/h) of perfusion rate corresponding to 0.15 (dynes/$\textrm{cm}^2$) of shear stress. The production of scu-PA is also increased as shear stress was slowly increased, which is similar to natural human blood circulation. The tube reactor proves that there may be no limitation of oxygen supply by showing 1.0 (1/h) of oxygen transfer coefficient at steady state and this system yields much lower shear stress of 0.3(dynes/$\textrm{cm}^2$) than that of 5-8(dynes/$\textrm{cm}^2$) by conventional agitation systems.

• KSBB Journal
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• v.8 no.1
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• pp.17-22
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• 1993

$1.96{\times}10^{-5}$(IU/cell/hr) of specific scu-PA production rate was obtained from HEK cells in maintaining ca. $8{\times}10^{5}$(cells/ml) of maximum roll density at 10(ml/hr) of perfusion rate with recycling 20% serum free conditioned media. It can be compared to $4{\times}10^{6}$(cells/ml) of maximum cell density and $4.56{\times}10^{-4}$(IU/cell/hr) of specific production rate in cultivating cells with 1% serum containing medium. Thc conversion ratio of scu-PA to tc-UK increased up to 55% as the recycling ratio increased; however, recycling the used medium seemed to have least negative effect on cell growth. It also showed that the recycling process had definitive advantage of using serum free medium in perfusion cultivation of HEK cell line.

• KSBB Journal
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• v.7 no.2
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• pp.132-138
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• 1992

Sprial adaptation technique of conditioned media has been applied to cultivate human cell line which can not survive in a serum free mdium without adding any growth factors in basal medium Doubling time and scu-PA production from serum free adapted cells were 5 days and 890 (IU/mL), respectively in a T-flask, whose values were not much lower than the productivity of 1100(IU/mL) from 5% serum containing medium. It was required to use conditioned media for attaching cells on microcarriers when cells were inoculated into a spinner vessel. Then, cells could continuously grow in serum free medium with having specific growth rate of 0.106 (1/day) and specific scu-PA production rate of $1.58{\times}10_{-5}$(IU/cell/day) in batch cultivation.