Improved Bioprocess with CHO-hTSH Cells on Higher Microcarrier Concentration Provides Higher Overall Biomass and Productivity for rhTSH

Abstract

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 (°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 °C, 20% DO, and pH 7.2 and starting the rhTSH synthesis phase with 3 × 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 (∼80%) of fully covered MCs for a long period (5 days) and attain a high cell concentration (∼9 × 10(5) cells/mL). The novelty of the present study is represented by the establishment of cell culture conditions allowing us to produce ∼1.6 mg/L of rhTSH in an already suitable degree of purity. Batches of produced rhTSH were purified and showed biological activity.

Progress in fed-batch culture for recombinant protein production in CHO cells

“…Most of DO values in CHO cells are usually in the range of 20 to 50% (Fleischaker and Sinskey 1981 ). Ventini et al reported that the final recombinant thyroid-stimulating hormone (rTSH) titer was 1.6 mg/L when DO value was 20% in fed-batch culture mode (Ventini et al 2011 ). To improve the yield and quality of human IgG1 mAb, Mahé et al adjusted the DO value to 40%, and CHO cells expressing human IgG1 mAb were cultured in fed-batch culture supplied with Copper Acetate and Ferric Citrate.…”

Design and Biophysical Characterization of Poly (l-Lactic) Acid Microcarriers with and without Modification of Chitosan and Nanohydroxyapatite

“…Since the concept of microcarriers was first proposed by Van Wezel in 1967 [1], microcarriers with particle size in a certain micrometer range have received extensive attention and development in drug delivery, defect filling, and three-dimensional cell culture [2,3,4,5,6,7,8]. Compared to conventional monolayer cell culture and three-dimensional static culture with a porous scaffold, utilizing microcarriers can increase the specific surface area and achieve the uptake of cells attached on their curved surface within a short period of time.…”

Purificação e imunogenicidade da glicoproteína do vírus da raiva (RVGP) expressa pelos sistemas células S2 e Semliki Forest Virus.

“…Provavelmente os altos níveis de expressão de proteínas já atingidos e reportados na literatura se devem ao uso de promotores fortes e alto número de cópias de plasmídeos integrados ao genoma (BUCKINGHAM et al, 1996;MCCARROLL;KING, 1997 (VENTINI et al, , 2011 e concentração celular máxima entre 3 -5x10 7 cel/mL (BATISTA et al, 2011;PAMBOUKIAN et al, 2008; BATISTA et al, 2011;BOVO et al, 2008;DOS SANTOS et al, 2009;GALESI et al, 2008;SWIECH et al, 2008aSWIECH et al, , 2008bSWIECH et al, , 2008cYOKOMIZO et al, 2007 produzir uma poliproteína estrutural precursora (C-p62-6K-E1), que é clivada cotraducionalmente pela função de protease da proteína C, gerando a proteína C (capsídeo) e o complexo das glicoproteínas do envelope viral (p62, 6K e E1). A proteína C reconhece um sinal de encapsidação que esta na região codificadora para nsP2 e junto com o RNA positivo completo (42S) forma o nucleocapsídeo (KUJALA et al, 2001;LOPEZ et al, 1994;KIELIAN, 2000;SKOGING et al, 1996;SUOMALAINEN;GAROFF, 1992;ZHAO;GAROFF, 1992).…”