A consistent increase of ∼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 ∼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.