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

Abstract

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.

Recent Developments in Protein and Peptide Parenteral Delivery Approaches

“…Thyrogen is indicated for diagnosis and treatment of thyroid cancer with a multidose regimen owing to short-circulating half-life. To reduce dosing frequency and extend duration of action, PEGylation strategy was attempted [17]. This study demonstrated successful PEGylation of a cysteine-knot with higher-reaction yield (approximately 85%) for monoPEGylated rhTSH.…”

Structure‐Based Site‐Specific PEGylation of Fibroblast Growth Factor 2 Facilitates Rational Selection of Conjugate Sites

“…For example, a fully active, long‐acting PEGylated factor VIII (FVIII) for hemophilia A treatment was engineered by site‐specific PEGylation based on the domain structure of full‐length FVIII . Another example is mono‐PEGylated human thyroid stimulating hormone (TSH), which was designed based on a structural model of the TSH–TSH receptor complex . There is a growing awareness that avoidance of reduced bio‐activity of PEGylated products requires site‐specific PEGylation (such as N‐terminal, Thiol, or C‐terminal PEGylation), as well as rational and tailored PEGylation modification strategies based on the structure of the target proteins.…”

High molecular weight PEGylation of human pancreatic polypeptide at position 22 improves stability and reduces food intake in mice

“…Several previous investigations have reported a deterioration of target potency with increasing PEG mass (Gong et al , ; Bianchi et al , ; Qiu et al , ), which is thought to originate from PEG‐chain length‐dependent altered steric shielding (Mu et al , ). We found that PEGylation of hPP on lysine side‐chains at positions 22 and 30 using different precursor peptides (hPP 2–36 and [Q 34 ]hPP) also induced significant reductions in activity that correlated mostly with PEG size.…”