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Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo.

Abstract
Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic proteins. However, its broader application to proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the proteins linked to albumin. We hypothesized that the site-specific conjugation of albumin to a permissive site of a target protein will expand the utilities of albumin as a therapeutic activity extender to proteins with a complex structure. We show here the genetic incorporation of a non-natural amino acid (NNAA) followed by chemoselective albumin conjugation to prolong therapeutic activity in vivo. Urate oxidase (Uox), a therapeutic enzyme for treatment of hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized human serum albumin (HSA) through strain-promoted azide-alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL x h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting protein therapeutics with high potency and safety.
AuthorsSung In Lim, Young S Hahn, Inchan Kwon
JournalJournal of controlled release : official journal of the Controlled Release Society (J Control Release) Vol. 207 Pg. 93-100 (Jun 10 2015) ISSN: 1873-4995 [Electronic] Netherlands
PMID25862515 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
CopyrightCopyright © 2015 Elsevier B.V. All rights reserved.
Chemical References
  • ALB protein, human
  • Fungal Proteins
  • Recombinant Fusion Proteins
  • Serum Albumin
  • Urate Oxidase
  • Serum Albumin, Human
Topics
  • Animals
  • Area Under Curve
  • Aspergillus flavus (enzymology, genetics)
  • Drug Stability
  • Enzyme Stability
  • Female
  • Fungal Proteins (administration & dosage, biosynthesis, chemistry, genetics, pharmacokinetics)
  • Half-Life
  • Injections, Intravenous
  • Mice, Inbred C57BL
  • Protein Engineering
  • Recombinant Fusion Proteins (biosynthesis)
  • Serum Albumin (administration & dosage, biosynthesis, chemistry, genetics, pharmacokinetics)
  • Serum Albumin, Human
  • Urate Oxidase (administration & dosage, biosynthesis, chemistry, genetics, pharmacokinetics)

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