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