Maria Teresa C. P. Ribela

A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and β-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t_1/2) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition.

Human thyrotropin (hTSH) is a glycoprotein with three potential glycosylation sites: two in the α-subunit and one in the β-subunit. These sites are not always occupied and occupancy is frequently neglected in glycoprotein characterization, even though it is related to folding, trafficking, initiation of inflammation and host defense, as well as congenital disorders of glycosylation (CDG). For the first time N-glycoprofiling analysis was applied to the site-occupancy determination of two native pituitary hTSH, in comparison with three recombinant preparations of hTSH, a widely used biopharmaceutical. A single methodology provided the: (i) average N-glycan mass; (ii) mass fraction of each monosaccharide and of sulfate; and (iii) percent carbohydrate. The results indicate that the occupancy (65%–87%) and carbohydrate mass (12%–19%) can be up to 34%–57% higher in recombinant hormones. The average glycan mass is 24% lower in pituitary hTSH and contains ~3-fold fewer moles of galactose (p < 0.005) and sialic acid (p < 0.01). One of the two native preparations, which had the smallest glycan mass together with the lowest occupancy and GalNAc, sulfate, Gal and sialic acid contents, also presented the lowest in vivo bioactivity and circulatory half-life. The methodology described, comparing a recombinant biopharmaceutical to its native equivalent, can be applied to any physiologically or clinical relevant glycoprotein.

Reversed-phase high performance liquid chromatography (RP-HPLC) was compared to the in vivo bioassay (BA) based on TSH-induced T₄ determination in mice. A linear relationship (BA_μg = 0.9790 RP-HPLC_μg − 0.052) with a highly significant correlation (r = 0.87; p < 0.001; n = 14 preparations) was found between these two methods. The mean difference between the bioactivity predicted from RP-HPLC data via this equation and the mean of the bioactivities obtained with the two methods for 11 other hTSH preparations was −2.01%, with a 95% confidence interval of −13.13% to +9.11%. This analysis included 5 totally or partially-degraded samples, indicating a useful correlation between the two determinations and, as expected, a higher sensitivity of the physical–chemical method. Interestingly, a commercial hTSH lot did not show any alteration 2 years post expiration date. These results demonstrate that RP-HPLC is a novel and viable alternative to the use of in vivo bioassays for hTSH potency determination, thus avoiding or reducing animal use.

The influence of sodium butyrate (NaBu) on the synthesis of recombinant human thyrotropin (r-hTSH) by CHO cells was investigated for the first time. A volumetric productivity of ~10 μg hTSH/mL was repeatedly obtained, with a 3.3-fold increase over a control culture carried out in the absence of NaBu. Since NaBu can induce CHO cell apoptosis and cell growth arrest, the increase in specific productivity was even higher, i.e., ca. 5-fold. Analysis of the N-glycan composition of r-hTSH obtained with the addition of NaBu to the culture medium showed an approximately 12 % increase in the amount of sialic acid, as well as in total carbohydrate, partly due to the increase in the site occupancy from 2.77 to 2.93 glycans per mole of hTSH. The two hormone preparations were characterized by N-glycan structural analysis, which showed that NaBu increased the bi-antennary structures by ca. 13 % while decreasing the tri-antennary structures by approximately the same amount. The in vivo biological activity and pharmacokinetic behavior (clearance) were found to be similar for the two hormone preparations.

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.