A CHO cell line, previously genetically modified by the introduction of rat alpha 2,6-sialyltransferase cDNA, generated for the first time a human-like sialylated recombinant hTSH (hlsr-hTSH) more similar to the native hormone, with 61% of alpha 2,3- and 39% of alpha 2,6-linked sialic acid residues. The best clone, when submitted to gene amplification with up to 8 mu M methotrexate, presented a secretion level of similar to 2 mu g hTSH/10(6) cells/day, useful for product purification and characterization. The relative molecular masses (M-r) of the heterodimer and of the alpha- and beta-subunits of purified hlsr-hTSH, determined by MALDI-TOF mass spectrometry, and the relative hydrophobicities, determined by RP-HPLC, were not remarkably different from those presented by two r-hTSH preparations secreted by normal CHO cells. Some differences were observed, though, in N-glycan composition, with more tri- and much more tetra-sialylated structures in hlsr-hTSH. When analyzed via an in vivo bioassay based on hTSH-induced T-4 release in mice, hlsr-hTSH was shown to be equipotent (p > 0.05) with the commercial preparation of r-hTSH (Thyrogen), and 1.6-fold more potent than native hTSH (p < 0.001). (C) 2009 Elsevier Inc. All rights reserved.