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 />
1305<br />
4.3×10 6 cells/ml in the control to 2.7×10 6 cells/<br />
ml. The activity <strong>of</strong> the rhFVIII was estimated to<br />
be 5.2% at hyperosmotic conditions when<br />
compared to 11.6% in the control. Accumulation<br />
<strong>of</strong> intracellular osmolytes such as Na + <strong>and</strong> Cl -<br />
ions cause instability <strong>of</strong> enzymes <strong>and</strong> RNA, <strong>and</strong><br />
consequently decrease cell density <strong>and</strong><br />
productivity (16).<br />
acetyl-CoA carboxylase involving<br />
gluconeogenesis <strong>and</strong> fatty acid biosynthesis; it<br />
can be considered as an effective parameter with<br />
regard to cell proliferation in serum-free media.<br />
Fig. 7. The rCHO cell morphology at different<br />
temperatures; (A) 30 o C <strong>and</strong> (B) 37 o C<br />
Effect <strong>of</strong> serum elimination on the rCHO<br />
density: Serum is normally enriched with<br />
precursors, growth factors, vitamins, <strong>and</strong>amino<br />
acids, which can remarkably increase<br />
mammalian cell proliferation. On the other h<strong>and</strong>,<br />
these ingredients cause problems in downstream<br />
processing <strong>of</strong> recombinant proteins. In addition,<br />
serum composition is variable resulting in a<br />
process with non-reproducibility. However,<br />
elimination <strong>of</strong> serum should be accompanied by<br />
adding appropriate substances, such as amino<br />
acids <strong>and</strong> vitamins. To eliminate serum from the<br />
culture medium, four amino acids as well as<br />
biotin were added <strong>and</strong> their effects on the rCHO<br />
cell viability were investigated. Figure 8 shows<br />
that approximately 80% <strong>of</strong> the rCHO cells were<br />
viable in presence <strong>of</strong> amino acids <strong>and</strong> biotin in<br />
serum-free medium, after 48 h <strong>of</strong> cultivation,<br />
whereas the rCHO cell viability was around 60%<br />
in the absence <strong>of</strong> serum <strong>and</strong> additives. So, these<br />
additives can be considered as suitable<br />
substitutes for serum in the rCHO cell culture<br />
medium. Since, biotin is a c<strong>of</strong>actor <strong>of</strong> a number<br />
<strong>of</strong> vital enzymes, e.g. pyruvate carboxylase <strong>and</strong><br />
Fig. 8. The effect <strong>of</strong> amino acids <strong>and</strong> biotin as additives<br />
on the rCHO cell density in serum-free medium<br />
Packed-bed bioreactor culture: Cell density in<br />
the packed-bed bioreactor reached 18×10 8 cells/<br />
l after 120 h <strong>of</strong> cultivation. The glucose<br />
concentration was depleted to 0.3 g/l at the end<br />
<strong>of</strong> cultivation. The time pr<strong>of</strong>ile <strong>of</strong> glucose<br />
consumption indicated that the lag phase in the<br />
bioreactor was approximately 18 h (Figure 9).<br />
Ammonium <strong>and</strong> lactate were also measured<br />
daily. The results showed that the ammonium <strong>and</strong><br />
lactate concentrations were at the inhibitory<br />
levels <strong>of</strong> 1.05 <strong>and</strong> 0.072 g/l at 120 h <strong>of</strong><br />
cultivation, respectively (Figure 9).<br />
Fig. 9. Time pr<strong>of</strong>ile in the packed-bed bioreactor<br />
culture, () glucose consumption, () ammonium<br />
formation, () lactate formation<br />
Shakibaie et al