Site-specific PEGylation of human thyroid stimulating hormone to prolong duration of action.
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.
Biologics R&D, Genzyme Corporation, A Sanofi Company, Framingham, MA 01701, USA. huawei.qiu@genzyme.com
Huawei
H
Qiu
Ekaterina
E
Boudanova
Anna
A
Park
Julie J
JJ
Bird
Denise M
DM
Honey
Christine
C
Zarazinski
Ben
B
Greene
Jonathan S
JS
Kingsbury
Susan
S
Boucher
Julie
J
Pollock
John M
JM
McPherson
Clark Q
CQ
Pan
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D003692:Delayed-Action Preparations / Q000008:administration & dosage / Q000737:chemistry; D005260:Female; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D011092:Polyethylene Glycols / Q000008:administration & dosage* / Q000737:chemistry*; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013972:Thyrotropin / Q000008:administration & dosage* / Q000737:chemistry* / Q000235:genetics; D013997:Time Factors
D003692:Delayed-Action Preparations; D011092:Polyethylene Glycols; D013972:Thyrotropin