| Author(s): |
Ventini, Daniella C.
[1, 2]
;
Damiani, Renata
[3]
;
Sousa, Alvaro P. B.
[1, 2]
;
de Oliveira, Joao E.
[3]
;
Peroni, Cibele N.
[3]
;
Ribela, Maria T. C. P.
[3]
;
Bartolini, Paolo
[3]
;
Tonso, Aldo
[2]
;
Soares, Carlos R. J.
[3]
;
Pereira, Carlos A.
[1, 2]
Total Authors: 10
|
| Affiliation: | [1] Inst Butantan, Lab Imunol Viral, Sao Paulo - Brazil
[2] Univ Sao Paulo, Lab Celulas Anim, Dept Engn Quim, Escola Politecn, BR-09500900 Sao Paulo - Brazil
[3] CNEN, IPEN, Ctr Biotecnol, Sao Paulo - Brazil
Total Affiliations: 3
|
Since the recombinant thyroid-stimulating hormone (rhTSH) is secreted by stably transfected Chinese hamster ovary (CHO-hTSH) cells, a bioprocess consisting of immobilizing the cells on a substrate allowing their multiplication is very suitable for rhTSH recovering from supernatants at relative high degree of purity. In addition, such a system has also the advantage of easily allowing delicate manipulations of culture medium replacement. In the present study, we show the development of a laboratory scale bioprocess protocol of CHO-hTSH cell cultures on cytodex microcarriers (MCs) in a 1 L bioreactor, for the preparation of rhTSH batches in view of structure/function studies. CHO-hTSH cells were cultivated on a fetal bovine serum supplemented medium during cell growth phase. For rhTSH synthesis phase, 75% of supernatant was replaced by animal protein-free medium every 24 h. Cell cultures were monitored for agitation (rpm), temperature (A degrees C), dissolved oxygen (% DO), pH, cell concentration, MCs coverage, glucose consumption, lactate production, and rhTSH expression. The results indicate that the amount of MCs in the culture and the cell concentration at the beginning of rhTSH synthesis phase were crucial parameters for improving the final rhTSH production. By cultivating the CHO-hTSH cells with an initial cell seeding of four cells/MC on 4 g/L of MCs with a repeated fed batch mode of operation at 40 rpm, 37 A degrees C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 x 10(6) cells/mL, we were able to supply the cultures with enough glucose, to maintain low levels of lactate, and to provide high percent (similar to 80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (similar to 9 x 10(5) cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to produce similar to 1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological activity. (AU) |