Influence of a reduced CO2 environment on the secretion yield, potency and N-glycan structures of recombinant thyrotropin from CHO cells.
A consistent increase of approximately 60% in the secretion yield of CHO-derived hTSH was observed by changing cell culture CO2 conditions from 5% CO2 to an air environment. The overall quality of the products obtained under both conditions was evaluated in comparison with a well-known biopharmaceutical (Thyrogen). The N-glycans identified were of the complex type, presenting di-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing approximately 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more di-antennary structures obtained in the absence of CO2 and 7-9% more tri-antennary structures in its presence. No remarkable difference in charge isomers was observed between the three preparations, the isoelectric focusing profiles showing six distinct bands in the 5.39-7.35 pI range. A considerably different distribution, with more forms in the acidic region, was observed, however, for two native pituitary preparations. All recombinant preparations showed a higher in vivo bioactivity when compared to native hTSH. Different production processes apparently do not greatly affect N-glycan structures, charge isomer distribution or bioactivity of CHO-derived hTSH.
Oliveira
João Ezequiel
JE
Biotechnology Department, IPEN-CNEN, Av. Prof. Lineu Prestes, 2242, Cidade Universitária, Sao Paulo 05508-900, Brazil.
Damiani
Renata
R
Vorauer-Uhl
Karola
K
Bartolini
Paolo
P
Ribela
Maria Teresa C P
MT
D000818:Animals; D016466:CHO Cells; D002245:Carbon Dioxide / Q000032:analysis*; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D015592:Immunoradiometric Assay; D007525:Isoelectric Focusing; D051379:Mice; D011134:Polysaccharides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013972:Thyrotropin / Q000096:biosynthesis*
D011134:Polysaccharides; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D013972:Thyrotropin