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Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1298 -1308 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1305 4.3×10 6 cells/ml in the control to 2.7×10 6 cells/ ml. The activity of the rhFVIII was estimated to be 5.2% at hyperosmotic conditions when compared to 11.6% in the control. Accumulation of intracellular osmolytes such as Na + and Cl - ions cause instability of enzymes and RNA, and consequently decrease cell density and productivity (16). acetyl-CoA carboxylase involving gluconeogenesis and fatty acid biosynthesis; it can be considered as an effective parameter with regard to cell proliferation in serum-free media. Fig. 7. The rCHO cell morphology at different temperatures; (A) 30 o C and (B) 37 o C Effect of serum elimination on the rCHO density: Serum is normally enriched with precursors, growth factors, vitamins, andamino acids, which can remarkably increase mammalian cell proliferation. On the other hand, these ingredients cause problems in downstream processing of recombinant proteins. In addition, serum composition is variable resulting in a process with non-reproducibility. However, elimination of serum should be accompanied by adding appropriate substances, such as amino acids and vitamins. To eliminate serum from the culture medium, four amino acids as well as biotin were added and their effects on the rCHO cell viability were investigated. Figure 8 shows that approximately 80% of the rCHO cells were viable in presence of amino acids and biotin in serum-free medium, after 48 h of cultivation, whereas the rCHO cell viability was around 60% in the absence of serum and additives. So, these additives can be considered as suitable substitutes for serum in the rCHO cell culture medium. Since, biotin is a cofactor of a number of vital enzymes, e.g. pyruvate carboxylase and Fig. 8. The effect of amino acids and biotin as additives on the rCHO cell density in serum-free medium Packed-bed bioreactor culture: Cell density in the packed-bed bioreactor reached 18×10 8 cells/ l after 120 h of cultivation. The glucose concentration was depleted to 0.3 g/l at the end of cultivation. The time profile of glucose consumption indicated that the lag phase in the bioreactor was approximately 18 h (Figure 9). Ammonium and lactate were also measured daily. The results showed that the ammonium and lactate concentrations were at the inhibitory levels of 1.05 and 0.072 g/l at 120 h of cultivation, respectively (Figure 9). Fig. 9. Time profile in the packed-bed bioreactor culture, () glucose consumption, () ammonium formation, () lactate formation Shakibaie et al
Current Trends in Biotechnology and Pharmacy Vol. 5 (3) 1298 -1308 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online) 1306 Activity of rhFVIII reached approximately 7% after 120 h of cultivation when compared to that of the pooled plasma, while this amount was 0% and 1.2% at 48 h and 96 h of cultivation, respectively. These results demonstrated that there is a direct relationship between protein production and cell density. Although there is no report on rhFVIII production in packed-bed bioreactors in the literature, but they have been employed for production of some recombinant pharmaceuticals (4). Trombopoietin was produced by the rCHO cells attached to polyester microcarriers in a packed-bed bioreactor. The maximum cell density and productivity of 2×10 7 cells/ml and 1.3-1.8 mg/l.d were obtained after 15 days, respectively (Cong et al. 2001). Because of the low stability of rhFVIII, the culture time period should be decreased. The results show that rhFVIII is biologically active during 120 h of cultivation when using enriched medium, in the packed bed bioreactor. Conclusion To our knowledge, no reports have been found in the literature on the physico-chemical culture conditions of recombinant CHO cells for improvement of rhFVIII production. The results of this study have demonstrated that medium supplemented with amino acids, precursors and glucose has a significant positive effect on the rCHO cell density and rhFVIII production, mainly because of the reduction of by-products formation. These data have provided useful information for the process development. Since rhFVIII is very sensitive to proteolytic degradation, culture time is an important factor in achieving an appropriate rhFVIII activity. Therefore, continuous packed-bed or perfusion cultures are suggested as suitable systems for production of rhFVIII in large-scale cultivation. Acknowledgments This work was financially supported in part by the International Center for Genetic Engineering and Biotechnology (ICGEB, Italy). The authors thank Dr. Parvin Shariati for her great assistance. References 1. Wurm, F.M. (2004). Production of recombinant therapeutics in cultivated mammalian cells. Nat Biotechnol, 22:1393- 1398. 2. Oh, D.J., Choi, S.K. and Changt, H.N. (1994). High density continuous cultures of hybridoma cell in a depth filter prefusion system. Biotechnol Bioengin, 44:895-901. 3. Kim, DY., Lee, J.C, Chang, H.N. and OH, D.J. (2005) Effects of supplementation of various medium components on Chinese Hamster Ovary cell cultures producing recombinant antibody. Cytotechnology, 47:37–49. 4. Cong, C., Chang, Y., Deng, J., Xiao, C. and Su, Z. (2001). A novel scale-up method for mammalian cell culture in packed-bed bioreactor. Biotechnol Lett, 23:881-885. 5. Sanfeliu, A. and Stephanopoulos, G. (1999). Effect of glutamine limitation on the death of attached Chinese Hamster Ovary cells. Biotechnol Bioengin, 64:47-53. 6. Landauer, K., Durrschmid, M., Klug, H., Wiederkum, S., Bluml, G. and Doblhoff- Dier,O. (2002). Detachment factors for enhanced carrier to carrier transfer of CHO cell lines on macroporous microcarriers. Cytotechnology, 39:37-45. 7. Lu, S., Sun, X. and Zhang, Y. (2005). Insight into metabolism of CHO cells at low glucose concentration on the basis of the determination of intracellular metabolites. Process Biochem, 40:1917–1921. 8. Sun, X. and Zhang, Y.(2004). Glutamine cannot support recombinant CHO cell Effect of culture conditions on rCHO cell growth
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(December 7-9, 2011) Venue: Karunya
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