full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
full issue - Association of Biotechnology and Pharmacy
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Current Trends in <strong>Biotechnology</strong> <strong>and</strong> <strong>Pharmacy</strong><br />
Vol. 5 (3) 1298 -1308 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)<br />
1298<br />
Improvement <strong>of</strong> rCHO Cell Density <strong>and</strong> Heterologous Protein<br />
Production: Effect <strong>of</strong> Culture Conditions<br />
M. Shakibaie 1,2 , F. Tab<strong>and</strong>eh 1* , A. Zomorodipour 3 , S. Ebrahimi 2 , F. Ataie 3 <strong>and</strong><br />
H. Habib Ghomi 1<br />
1<br />
Department <strong>of</strong> Industrial <strong>and</strong> Environmental <strong>Biotechnology</strong>, National Institute <strong>of</strong> Genetic Engineering <strong>and</strong><br />
<strong>Biotechnology</strong>, P.O. Box: 14965-161, Tehran, Iran<br />
2<br />
<strong>Biotechnology</strong> Group, Chemical Engineering Faculty, Sah<strong>and</strong> University <strong>of</strong> Technology, Tabriz, Iran<br />
3<br />
Department <strong>of</strong> Molecular Genetics,<br />
National Institute <strong>of</strong> Genetic Engineering <strong>and</strong> <strong>Biotechnology</strong>, P.O. Box: 14965-161, Tehran, Iran<br />
* For Correspondence - taban_f@nigeb.ac.ir<br />
Abstract<br />
Physico-chemical parameters have a great<br />
impact on mammalian cell growth. In this study,<br />
the effects <strong>of</strong> a number <strong>of</strong> amino acids,<br />
precursors, serum, glucose, osmolality <strong>and</strong><br />
temperature on the recombinant Chinese hamster<br />
ovary (rCHO) cells producing the recombinant<br />
human coagulation factor VIII (rhFVIII) were<br />
investigated, so as to improve the rCHO cell<br />
density in the packed-bed bioreactor. The results<br />
showed that while the concentration <strong>of</strong><br />
ammonium, as the main by-product, increased<br />
by more than 6mM, the rCHO cell density<br />
decreased significantly. On the other h<strong>and</strong>,<br />
adding certain amino acids <strong>and</strong> precursors<br />
mitigated the stress caused by ammonium <strong>and</strong><br />
consequently increased cell density. Proline <strong>and</strong><br />
glycine increased cell density up to<br />
approximately 28% in comparison with the<br />
negative control. Nucleic acids, as the main<br />
precursors, had a significant impact on rCHO<br />
cell growth, which increased by up to 20%.<br />
Higher amounts <strong>of</strong> glucose (3.5 g/l) supplied the<br />
carbon <strong>and</strong> energy requirements <strong>of</strong> the cells,<br />
inhibited by-products formation <strong>and</strong> sequentially<br />
increased heterologous protein production. The<br />
precursors allowed a 14% improvement in<br />
rhFVIII activity at high glucose concentrations,<br />
which was 1.5 fold greater than the negative<br />
control. Both hyperosmotic pressure <strong>and</strong><br />
temperature downshifts had remarkable adverse<br />
effects on the rCHO cell density,; being reduced<br />
to 2.7×10 6 cells/ml. The acquired suitable culture<br />
conditions were applied to the packed-bed<br />
bioreactor, <strong>and</strong> consequently an rCHO cell<br />
density <strong>of</strong> 18×10 8 cells/l was achieved during<br />
120 h <strong>of</strong> cultivation. The results <strong>of</strong> this study<br />
indicated that the rCHO cells essentially need<br />
supplementary materials to deal with<br />
inappropriate culture conditions; especially in a<br />
serum-free medium.<br />
Keywords: Factor VIII, Packed-bed bioreactor,<br />
Recombinant CHO cells, Serum-free medium.<br />
Introduction<br />
Mammalian cell cultures are necessary for<br />
production <strong>of</strong> glycosylated recombinant<br />
pharmaceutical proteins (1). Generally,<br />
mammalian cells have low productivity,<br />
Effect <strong>of</strong> culture conditions on rCHO cell growth