key: cord-0038808-l4dyowo1
authors: Lee, Chang-Jin; Kim, Cheol-Min; Park, Jeong-Nam; Jeong, Yeon-Ho
title: The improvement of porcine epidemic diarrhea vaccine production through in situ removal of ammonium ions by zeolite
date: 2008-10-16
journal: J Biotechnol
DOI: 10.1016/j.jbiotec.2008.07.289
sha: 703d99a1e7443b2ef48a334fe6b46145766a8d07
doc_id: 38808
cord_uid: l4dyowo1

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Abstracts / Journal of Biotechnology 136S (2008) S130-S139 teins. A fusion protein, human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) was strongly induced by sugar starvation using rice alpha-amylase 3D (RAmy3D) promoter in transgenic rice cell suspension cultures. Nevertheless, there are some considerations which this inducible promoter system caused cell death because of sugar depletion during production phase. Delay of the production due to existence of the residual sugar was an indispensable problem during the induction period by medium exchange in bioreactor. In addition, loss of productivity associated with scale-up in flasks or bioreactors was related with hydrodynamic stress. In this study, the effects of inoculation methods and flask size on production of hCTLA4Ig were confirmed. Different flask sizes (0.1, 0.25, 0.5, 1, 2 and 5 L) and inoculation methods (suspension and filtered cells) were applied to investigate their effects during production phase in Erlenmeyer flasks. It was found that the volume ratio of spent medium containing residual sugar and settled cells, and flask size specifically influenced hCTLA4Ig expression pattern, cell viability and protease activity. In conclusion, it was essential to minimize the residual sugar in suspensions and hydrodynamic stress by scale-up for the enhancement of the production with RAmy3D promoter system. Porcine epidemic diarrhea virus (PEDV) is coronavirus and a causative agent for diarrhea in pigs, particularly in neonates. The propagation of the PEDV in cell culture was developed in 1988. PED disease vaccine is produced from Vero cells by using lively attenuated virus strain (Hofmann and Wyler, 1988) . Industrially important viruses such as viral vaccines have been typically produced using anchorage-dependent cell lines in host cell culture systems (Lee et al., 1995) . The accumulation of ammonium ion in culture media is inevitable because ammonium ion is excreted as a byproduct of glutamine metabolism which is essential to mammalian cells. The inhibitory effect of ammonium ion is common to most animal cells. Ammonium ion inhibits not only cell growth but also product formation such as interferon and vaccine. Therefore, high cell density and high vaccine productivity could be achieved if ammonium ions were removed from the media as soon as they formed. In this study, the PEDV vaccine was produced by immobilized Vero cell in spinner flask coupled with in situ removal of ammonium ion. A synthetic zeolite of membrane type was used as an ammonia adsorbents (Park et al., 1998) .

The optimum operation variables such as multiplicity of infection (MOI), infection time, and harvest time was investigated before the in situ removal system was applied. The optimum operation variables for PEDV vaccine production in a spinner culture were MOI of 0.01 cell −1 , infection time of 72 h and harvest time of 48 h. The optimum time for zeolite addition to 100 ml spinner flask in terms of volumetric virus productivity and specific virus productivity were 72 h and 48 h, respectively. The volumetric virus productivity with zeolite treatment system was 2.6 times higher than that without zeolite treatment system in the media supplemented with 2.5 mM glutamine. The results showed that the integrated cell culture system coupled with immobilized cell culture and in situ removal of ammonium ion was very effective for the production of PEDV vaccine.

Propagation of the virus of porcine epidemic diarrhea in cell culture

Production of Newcastle disease virus using Vero cell culture. Kor

Development of an immobilized adsorbent for in situ removal of ammonium ion from animal cell culture media and its application to animal cell culture system: application to cell culture system. Kor

Iduronate 2-sulphatase (IDS) is a lysosomal enzyme involved in the sequential degradation of the glycosaminoglycans heparan, sulphate and dermaran sulphate. Mucopolysaccharidosis type disease (MPS , Hunter syndrome) is caused by a deficiency of IDS (Timms and Bondeson, 1997). Thus industrial production of IDS by recombinant CHO cells is highly required for the treatment of Hunter syndrome. For high-level expression of foreign proteins, sodium butyrate has been widely used as an additive in recombinant CHO cell cultures (Sung and Lee, 2005) . Also, recombinant protein yields can be increased significantly by lowering the culture temperature (Yoon et al., 2006) . In this study, the effect of sodium butyrate on the growth of recombinant CHO cells and on the IDS productivity were investigated in serum-free suspension culture. Cell culture with 3-10 mM sodium butyrate was known to arrest cell growth and to increase specific IDS productivity. The optimum concentration of sodium butyrate was found 5 mM and optimum addition time was after 2days in the batch culture at 37 • C. The effect of temperature on the IDS productivity were evaluated by cultivating IDS producing recombinant CHO cells with addition of sodium butyrate in exponential phase at 37 • C, 32 • C, and temperature shift of 37 • C to 32 • C. The optimum operating temperature profile was found to be the temperature shift of 37 • C to 32 • C, because higher