Site-Specific PEGylation of Human Thyroid Stimulating Hormone to Prolong Duration of Action

• Published in: Bioconjugate chemistry Vol. 24; no. 3; pp. 408 - 418
• Main Authors: Qiu, Huawei, Boudanova, Ekaterina, Park, Anna, Bird, Julie J, Honey, Denise M, Zarazinski, Christine, Greene, Ben, Kingsbury, Jonathan S, Boucher, Susan, Pollock, Julie, McPherson, John M, Pan, Clark Q
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 20.03.2013
• Subjects: Amino Acid Sequence; Animals; Binding sites; Binding Sites - drug effects; Binding Sites - physiology; Delayed-Action Preparations - administration & dosage; Delayed-Action Preparations - chemistry; Endocrine therapy; Female; Gene expression; Humans; Male; Molecular Sequence Data; Mutation; Oncology; Pharmacology; Polyethylene Glycols - administration & dosage; Polyethylene Glycols - chemistry; Protein Binding - drug effects; Protein Binding - physiology; Rats; Rats, Sprague-Dawley; Thyroid cancer; Thyrotropin - administration & dosage; Thyrotropin - chemistry; Thyrotropin - genetics; Time Factors
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc300519h

Recombinant human thyroid stimulating hormone (rhTSH or Thyrogen) has been approved for thyroid cancer diagnostics and treatment under a multidose regimen due to its short circulating half-life. To reduce dosing frequency, PEGylation strategies were explored to increase the duration of action of rhTSH. Lysine and N-terminal PEGylation resulted in heterogeneous product profiles with 40% or lower reaction yields of monoPEGylated products. Eleven cysteine mutants were designed based on a structure model of the TSH-TSH receptor (TSHR) complex to create unique conjugation sites on both α and β subunits for site-specific conjugation. Sequential screening of mutant expression level, oligomerization tendency, and conjugation efficiency resulted in the identification of the αG22C rhTSH mutant for stable expression and scale-up PEGylation. The introduced cysteine in the αG22C rhTSH mutant was partially blocked when isolated from conditioned media and could only be effectively PEGylated after mild reduction with cysteine. This produced a higher reaction yield, ∼85%, for the monoPEGylated product. Although the mutation had no effect on receptor binding, PEGylation of αG22C rhTSH led to a PEG size-dependent decrease in receptor binding. Nevertheless, the 40 kDa PEG αG22C rhTSH showed a prolonged duration of action compared to rhTSH in a rat pharmacodynamics model. Reverse-phase HPLC and N-terminal sequencing experiments confirmed site-specific modification at the engineered Cys 22 position on the α-subunit. This work is another demonstration of successful PEGylation of a cysteine-knot protein by an engineered cysteine mutation.