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 />
1302<br />
Ammonium concentration (mg/l)<br />
Lactate concentration (mg/l)<br />
Time (h)<br />
Fig.2. Time pr<strong>of</strong>ile <strong>of</strong> ammonium formation at<br />
different glucose concentrations; 1.2 g/l (), 3.5 g/l<br />
() <strong>and</strong> 7 g/l ()<br />
Time (h)<br />
Fig. 3. Time pr<strong>of</strong>ile <strong>of</strong> lactate formation at different<br />
glucose concentrations; 1.2 g/l (), 3.5 g/l () <strong>and</strong> 7<br />
g/l ()<br />
Glutamine is another carbon <strong>and</strong> main nitrogen<br />
source for rCHO cell metabolism. Glutamine is<br />
not essential for rCHO cells, which have<br />
glutamine synthetase that catalyzes the following<br />
reaction (28):<br />
L-glutamate + NH 4<br />
+<br />
+ ATP < > L-glutamine +<br />
ADP + Pi + H +<br />
ATP can be generated if glutamine is<br />
consumed, which commonly occurs at low<br />
glucose concentrations in order to replenish the<br />
energy deficit <strong>and</strong> consequently produce<br />
ammonium. As shown in Figure 3, there is no<br />
significant difference in lactate formation at<br />
various glucose concentrations up to 48 h. The<br />
rCHO cells then consume lactate as carbon<br />
source at the lower glucose concentration. So,<br />
detoxification <strong>of</strong> lactate can occur under glucose<br />
limitation. While the glucose concentration<br />
increased, there was no need to consume lactate<br />
for replenishing the energy deficit <strong>and</strong> the lactate<br />
concentration was not highly decreased.<br />
The effect <strong>of</strong> glucose concentration on<br />
recombinant protein production is shown in Table<br />
1. Activity <strong>of</strong> the recombinant hFVIII was<br />
increased to 8.4%when glucose concentrations<br />
were increased to 3.5 g/l, <strong>and</strong> then dropped <strong>of</strong>f<br />
to 4.2 % when glucose concentration reached 7<br />
g/l. The increase in activity may be due to the<br />
increased cell density at 3.5 g/l <strong>of</strong> glucose in<br />
culture medium. Another possible reason that can<br />
be attributed to the glucose concentration effect<br />
is ATP content <strong>of</strong> the cell which increases when<br />
glucose levels are appropriately high. In fact the<br />
chaperon immunoglobulin-binding protein (BiP)<br />
has been introduced as the main barrier in the<br />
secretion <strong>of</strong> rhFVIII. Release <strong>of</strong> BiP from<br />
rhFVIII depends on the intracellular content <strong>of</strong><br />
ATP (29,30).<br />
Table 1. Activity <strong>of</strong> rhFVIII at different<br />
concentrations <strong>of</strong> glucose<br />
Glucose concentration (g/l). rhFVIII activity (%)<br />
1.2 6.1<br />
3.5 8.4<br />
7.0 4.2<br />
Mitigation <strong>of</strong> ammonium stress by amino acid<br />
addition: Ammonium inhibits cell growth <strong>and</strong><br />
protein production as mentioned above. The<br />
critical level <strong>of</strong> ammonium inhibiting cell growth<br />
has been reported as 10 mM (14). Figure 4<br />
indicates that the rCHO cell density significantly<br />
decreased to 3.4×10 6 cells/ml at 4-6 mM NH 4<br />
Cl,<br />
Effect <strong>of</strong> culture conditions on rCHO cell growth