Human prolactin (hPRL) is a polypeptide hormone occurring in the non-glycosylated (NG-hPRL) and glycosylated (G-hPRL) forms, with MM of approximately 23 and 25kDa, respectively. It has a single, partially occupied N-glycosylation site located at Asn-31, which makes it a particularly simple and interesting model for glycosylation studies. The bioactivity of G-hPRL is lower than that of NG-hPRL (by ca. 4-fold) and its physiological function is not clear. However, carbohydrate moieties generally play important roles in the biosynthesis, secretion, biological activity, and plasma survival of glycohormones and can vary depending on the host cell. The main objective of this study was to determine the N-glycan structures present in native, pituitary G-hPRL and compare them with those present in the recombinant hormone. To obtain recombinant G-hPRL, genetically modified Chinese hamster ovary cells (CHO), adapted to growth in suspension, were treated with cycloheximide, thus increasing the glycosylation site occupancy from 5.5% to 38.3%, thereby facilitating G-hPRL purification. CHO cell-derived G-hPRL (CHO-G-hPRL) was compared to pituitary G-hPRL (pit-G-hPRL) especially with regard to N-glycoprofiling. Among the main differences found in the pituitary sample were an extremely low presence of sialylated (1.7%) and a high percentage of sulfated (74.0%) and of fucosylated (90.5%) glycans. A ∼6-fold lower in vitro bioactivity and a higher clearance rate in mice were also found for pit-G-hPRL versus CHO-G-hPRL. N-Glycan profiling proved to be a useful and accurate methodology also for MM and carbohydrate content determination for the two G-hPRL preparations, in good agreement with the values obtained directly via MALDI-TOF-MS.
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.
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