• KSBB Journal
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• 제13권3호
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• pp.231-237
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• 1998

세계보건기구 (WHO)가 백신 생산에 권장하고 있는 표준세포 주인 Vero 세포에 약독화 일본뇌염바이러스인 SA14-14-2 ( (PDK)를 연속 계대배양을 통해 적응(adaptation)시켜, tIter가 $10^7$pfu/mL을 넘는 SA-14-14-2(Vero)을 분리하였다 바이러스 배양 최적온도는 $35^{\circ}C$이며, T -flask에서 배양된 바이러스의 최고 tIter는 감염 후 4일째에 $4\times10^7$ pfu/mL로 관찰되었다. 또한 무혈청배지에서도 바이러스 증식이 활발하여 2% 혈청이 보충된 정우와 거의 비슷한 바이라스 tIter를 보였다. 바이러스 대량 배양을 위해 roller bottle culture와 미 립 담체 플 이용한 spinner flask culture 가능성에 대하여 고찰하였다 바이러스 감염을 위한 미립담체에서의 Vera cell monolayer는 초기 세포 농도 $4\times10$ cells/mL로 접종하여 50 rpm에서 7일간 배양하여 얻을 수 있었다. 바이러스의 roller bottle 배양이 spinner flask 배양보다 바이러스 tIter변에서 2배 내지 3배 높 았고, $10^7$pfu/mL을 넘는 배양 기간도 하루 죄었다. 하지만 두 배양 방법 모두 T -flask 배양에서와 같이 무혈청 배지를 사용 하여도 바이라스 증식이 활발했고, 최고조의 tIter를 보이는 배 양기간은 감염 후 2일째로써 T -flask 배양에서 보다 2일 빨랐다. Roller bottle culture의 경우, 감염 후 3일부터 17일까지 2 일 간격으로 배양액을 무혈청 EMEM으로 100% 교체하면서 매 양을 지속한 결과 3일부터 9일까지 $10^7$pfu/mL을 념는 tIter가 유지되는 것이 확인되어 바이러스의 multi-harvest가 가능한 것 로 고찰되었다. 상기의 결과는 생산성 면에서 매우 유리한 결 과로 제품의 생산 단가플 낮추고 작업 노력을 절감하는 기대 효 과가 클 것으로 예측된다.

• 대한수의학회지
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• 제26권2호
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• pp.259-264
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• 1986

Microcarrier culture technics are widely used for the massive production of the vertebrate animal cells. In this study, attempt was made to establish the microcarrier cell culture system using Cytodex. Various factors affecting the growth of cells on microcarrier culture were also discussed. In conclusion, the yield of cells in microcarrier culture was several times greater than those in roller bottle and flask culture methods, based on the volume of culture media.

• KSBB Journal
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• 제13권3호
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• pp.295-302
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• 1998

Human Erythropoietin (EPO) gene is cloned in quail fibrosarcoma cell, QT35. Because molecular weight of EPO is similar to that of serum albumin, cell culture with serum containing medium makes purification of EPO very difficult. Using fractional factorial study, we have developed serum free medium for the recombinant QT35 cell lines, QT N4D4 and QT SY-IMP, which have cloned EPO with glutamine synthetase (GS) gene amplification system and with puromycin selective marker, respectively. Among the seven frequently used medium components, fibronectin, BSA, and EGF were the most important for EPO production. However, sufficient fibronectin supplement to the medium did not make any good attachment of QT35 to culture plate over 3 days. Therefore, to maximize EPO production, we attempted a medium-shift at confluence from serum containing medium to serum free medium(QT SFM6). Using the medium-shift protocol with QT SFM6, nearly the same productivity of EPO was achieved comparing with that without medium-shift. This result was true in both QT35 cell lines in three types of culture, i.e. T flask, microcarrier and roller bottle cultures.

• Journal of Microbiology and Biotechnology
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• 제9권4호
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• pp.386-392
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• 1999

A large-scale culture of hepatitis A virus in human diploid MRC-5 cells was conducted. In a roller bottle culture, the virus was grown to a maximum titer in 3 weeks after infection. Over 95％ of the cell-associated virus was excreted after culturing the infected cells in suspension media without fetal bovine serum for 3 days. The cultured virus was inactivated with formalin, concentrated by ultrafiltration, and partially purified by ultracentrifugation in a non-ionic gradient medium of Renocal. Two separate peak fractions showing high anti-HAY ELISA titer were pooled and about 40％ of HAV antigen was recovered by this purification procedure. Of the partially purified vaccine, the protein pattern in SDS-PAGE and immunogenicity in mice were compared with a commercial HAV vaccine. In SDS-PAGE, the purified vaccine in this study and the commercial vaccine showed almost the same protein pattern. The seroconversion rate of the purified vaccine in mice was not different from that of the commercial vaccine. Therefore, we could prepare a good grade of HAV vaccine by a simple purification procedure although the purification itself was not completed.

• Biotechnology and Bioprocess Engineering:BBE
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• 제5권2호
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• pp.118-122
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• 2000

A comlementary call line CR2 is curretly used to propagte the Disabled Infectious Single Cycle Herpes Simplex Virus Typee2 (DISC HSV-2) on a small Iaboratory scale upto 15 L. These cultures are initiated by passaging the cells from roller bottle cultures. Whilst this is suitable for the laboratory scale it is totally impractical for use in seeding an industrial manufacturing scaled version of the culture. It is paramount to have a robust system for passaging cells from a small microcarrierier culture system to a larger one by a serial subculturing regime. Here we report on the successes we have had in our laboratory in scaling up out production system for the DISC HSV-2 from small 1-L cultures to a 50-L vessel with the maintenance of the viral productivity. Ease of use, reproducibility and the need to minimise overall production time were factors which were taken into consideration whils developing our procedures. We were aware of the need to keep a production train simple and as short as possible as this was the amall scale study for an envisaged manufacturing process.

• Archives of Pharmacal Research
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• 제21권5호
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• pp.521-526
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• 1998

The physicochmical properties of recombinant hepatitis B surface antigen (r-HBsAg), which was expressed in C127 mammalian cell were studied. Using roller bottle culture in DMEM supplemented with fetal bovine serum, 10-15 mg/L of r-HBsAg was produced with about 31% of purification yield. The purity of r-HBsAg by HPLC was 99.8% and electron microscopic examination showed homogeneous spherical particle with 22 nm in diameter, a morphological characteristic of HBsAg. The density of r-HBsAg by CsCI density gradient method was 1.19g/ml and the isoelectric point by Mono $P^{TM}$ HR 5/20 column was 4.6. The analysis of subunit protein pattern using SDS-PAGE followed by scanning densitometry gave 81.3% of S protein and 18.7% of pre-S protein. fluorophore-assisted-carbohydrate-electrophoresis analysis showed the relative amount of carbohydrate to protein was 1.7% and it smajr component was N-acetyl glucosamine, which was about 39% of total carbohydrate. The relative amount of lipid to protein determined by vanillin phosphoric acid method was 32.5% and its major component was phospholipid, which was about 70% of total lipid. The physicochemical properties of C127 mammalian cell-derved r-HBsAg are similar to those of p-HBsAg, suggesting that the r-HBsAg can be used in developing a new preventive vaccine against hepatitis B.