full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 5 (3) 1298 -1308 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)
1304
Cell density 10 6 cells/ml
Fig. 5. The rCHO cell density under ammonium stress at
low glucose concentrations, 1.2 g/l (black bars) and high
glucose concentrations, 3.5 g/l (white bars)
Effect of precursors on the rCHO cell density
and protein production: The rCHO cell growth
was promoted in the presence of the precursors.
Cell density increased by up to 5% and 20%
(ANOVA, p< 0.05) in comparison with the
control cultures, at low and high glucose
concentrations, respectively. The maximum cell
density of 1.2×10 7 cells/ml was achieved in the
presence of the precursors at high glucose
concentration. Ammonium formation also
decreased, when precursors were added to the
medium (Figure 6). The rhFVIII activities were
obtained as 10.58% and 14.02% at low and high
glucose concentration, as compared to the
negative control showing activities of 6.4% and
9.19%, respectively. dNTPs as precursors are
converted to glutamate in the metabolic
pathways. The consumption of glutamate forms
lesser amounts of ammonium than that of
glutamine. Furthermore, the precursors are
imported into the TCA cycle and improve cell
energetics. Another possible reason that can be
attributed to the nucleic acids is stoping the
elongation of RNA polymerase while the
interacellular concentration of nucleic acids
decreases (32).
Effect of temperature and osmolality on cell
density and rFVIII production: The rCHO cell
densities were 2.7×10 6 and 3.7×10 6 cells/ml at
30 o C and 37 o C, respectively. The activity of
Ammonium concentration (mg/l) Ammonium concentration (mg/l)
Fig. 6. Time profile of ammonium in the presence () and
absence ( ) of precursors at (A) low glucose
concentrations; 1.2 g/l and (B) high glucose concentrations;
3.5 g/l
rhFVIII increased 2.2-fold when temperature was
increased from 30 o C to 37 o C. Different cell lines
exhibit various reactions during their growth and
heterologous protein production in response to
temperature up- or down-shift. Since rhFVIII is
constitutively expressed in the rCHO cell line,
its production depends directly on cell density.
Figure 7 depicts the morphology of the rCHO
cells in response to a temperature downshift. The
proliferation of the rCHO cells was reduced at
30 o C and cell’s morphology changed to triangular
shapes, while the CHO cells at 37 o C are usually
spindle shaped. It has been reported that when
temperature is downshifted to 30 o C, a cell cycle
inhibitory protein named p27/KIP is overexpressed,
which has a negatively regulates
cyclin dependent kinases (17,18).
Hyperosmolality had negative effects on
the rCHO cell density, which decreased from
Effect of culture conditions on rCHO cell growth