Carbohydrate-mediated polyethylene glycol conjugation of TSH improves its pharmacological properties

Thyrogen (thyrotropin alfa for injection), recombinant human TSH (rhTSH), has been successfully used to enhance diagnostic radioiodine scanning and thyroglobulin testing in the follow-up of patients with thyroid cancer and as an adjunctive treatment for radioiodine thyroid remnant ablation. However, the short half-life of rhTSH in the circulation requires a multidose regimen. We developed novel sialic acid-mediated and galactose-mediated conjugation chemistries for targeting polyethylene glycol (PEG) to the three N-linked glycosylation sites on the protein, to prolong plasma half-life by eliminating kidney filtration and potential carbohydrate-mediated clearance. Conjugates of different PEG sizes and copy numbers were screened for reaction yield, TSH receptor binding, and murine phamacokinetics/pharmacodynamics studies. The best performing of these products, a 40-kDa mono-PEGylated sialic acid-mediated conjugate, exhibited a 3.5-fold longer duration of action than rhTSH in rats, as a 5-fold lower affinity was more than compensated by a 23-fold extension of circulation half-life. Biochemical characterization confirmed conjugation through the sialic acids. Correlation of PEG distribution on the three N-linked glycosylation sites and the PEG effect on receptor binding supported the previously reported structure-function relationship of rhTSH glycosylation. This long-acting rhTSH has the potential to significantly improve patient convenience and provider flexibility while reducing potential side effects associated with a sudden elevation of serum TSH.