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 />
1304<br />
Cell density 10 6 cells/ml<br />
Fig. 5. The rCHO cell density under ammonium stress at<br />
low glucose concentrations, 1.2 g/l (black bars) <strong>and</strong> high<br />
glucose concentrations, 3.5 g/l (white bars)<br />
Effect <strong>of</strong> precursors on the rCHO cell density<br />
<strong>and</strong> protein production: The rCHO cell growth<br />
was promoted in the presence <strong>of</strong> the precursors.<br />
Cell density increased by up to 5% <strong>and</strong> 20%<br />
(ANOVA, p< 0.05) in comparison with the<br />
control cultures, at low <strong>and</strong> high glucose<br />
concentrations, respectively. The maximum cell<br />
density <strong>of</strong> 1.2×10 7 cells/ml was achieved in the<br />
presence <strong>of</strong> the precursors at high glucose<br />
concentration. Ammonium formation also<br />
decreased, when precursors were added to the<br />
medium (Figure 6). The rhFVIII activities were<br />
obtained as 10.58% <strong>and</strong> 14.02% at low <strong>and</strong> high<br />
glucose concentration, as compared to the<br />
negative control showing activities <strong>of</strong> 6.4% <strong>and</strong><br />
9.19%, respectively. dNTPs as precursors are<br />
converted to glutamate in the metabolic<br />
pathways. The consumption <strong>of</strong> glutamate forms<br />
lesser amounts <strong>of</strong> ammonium than that <strong>of</strong><br />
glutamine. Furthermore, the precursors are<br />
imported into the TCA cycle <strong>and</strong> improve cell<br />
energetics. Another possible reason that can be<br />
attributed to the nucleic acids is stoping the<br />
elongation <strong>of</strong> RNA polymerase while the<br />
interacellular concentration <strong>of</strong> nucleic acids<br />
decreases (32).<br />
Effect <strong>of</strong> temperature <strong>and</strong> osmolality on cell<br />
density <strong>and</strong> rFVIII production: The rCHO cell<br />
densities were 2.7×10 6 <strong>and</strong> 3.7×10 6 cells/ml at<br />
30 o C <strong>and</strong> 37 o C, respectively. The activity <strong>of</strong><br />
Ammonium concentration (mg/l) Ammonium concentration (mg/l)<br />
Fig. 6. Time pr<strong>of</strong>ile <strong>of</strong> ammonium in the presence () <strong>and</strong><br />
absence ( ) <strong>of</strong> precursors at (A) low glucose<br />
concentrations; 1.2 g/l <strong>and</strong> (B) high glucose concentrations;<br />
3.5 g/l<br />
rhFVIII increased 2.2-fold when temperature was<br />
increased from 30 o C to 37 o C. Different cell lines<br />
exhibit various reactions during their growth <strong>and</strong><br />
heterologous protein production in response to<br />
temperature up- or down-shift. Since rhFVIII is<br />
constitutively expressed in the rCHO cell line,<br />
its production depends directly on cell density.<br />
Figure 7 depicts the morphology <strong>of</strong> the rCHO<br />
cells in response to a temperature downshift. The<br />
proliferation <strong>of</strong> the rCHO cells was reduced at<br />
30 o C <strong>and</strong> cell’s morphology changed to triangular<br />
shapes, while the CHO cells at 37 o C are usually<br />
spindle shaped. It has been reported that when<br />
temperature is downshifted to 30 o C, a cell cycle<br />
inhibitory protein named p27/KIP is overexpressed,<br />
which has a negatively regulates<br />
cyclin dependent kinases (17,18).<br />
Hyperosmolality had negative effects on<br />
the rCHO cell density, which decreased from<br />
Effect <strong>of</strong> culture conditions on rCHO cell growth