Evidence for Metabolic Cleavage of a PEGylated Protein in Vivo Using Multiple Analytical Methodologies

Abstract

PEGylation of therapeutic proteins is commonly used to extend half-lives and to reduce immunogenicity. However, reports of antibodies toward PEGylated proteins and of poly(ethylene glycol) (PEG) accumulation suggest that efficacy and safety concerns may arise. To understand the relationship among the pharmacology, immunogenicity, and toxicology of PEGylated proteins, we require knowledge of the disposition and metabolic fate of both the drug and the polymer moieties. The analysis of PEG by standard spectrophotometric or mass spectrometric techniques is problematic. Consequently, we have examined and compared two independent analytical approaches, based on gel electrophoresis and nuclear magnetic resonance (NMR) spectroscopy, to determine the biological fate of a model PEGylated protein, 40KPEG-insulin, within a rat model. Both immunoblotting with an antibody to PEG and NMR analyses (LOD 0.5 μg/mL for both assays) indicated that the PEG moiety remained detectable for several weeks in both serum and urine following intravenous administration of 40KPEG-insulin (4 mg/kg). In contrast, Western blotting with anti-insulin IgG indicated that the terminal half-life of the insulin moiety was far shorter than that of the PEG, providing clear evidence of conjugate cleavage. The application of combined analytical techniques in this way thus allows simultaneous independent monitoring of both protein and polymer elements of a PEGylated molecule. These methodologies also provide direct evidence for cleavage and definition of the chemical species present in biological fluids which may have toxicological consequences due to unconjugated PEG accumulation or immunogenic recognition of the uncoupled protein.

XTEN-Annexin A5: XTEN Allows Complete Expression of Long-Circulating Protein-Based Imaging Probes as Recombinant Alternative to PEGylation

“…Last, there are increasing safety concerns about PEGylation. Recently, it has been reported that PEGylated protein complexes may dissociate in vivo, resulting in free PEG moieties circulating in the bloodstream and tissues (5). This finding raises questions about potential long-term toxicity because PEG is nonbiodegradable (26).…”

Effects of pharmaceutical PEGylation on drug metabolism and its clinical concerns

“…Decreased activity could be compensated by the improved pharmacokinetic properties [33]. Although PEGylation has a prominent advantage of reducing the metabolism of PEGylated bioactives, the conjugates still experience slow degradation by protease or related enzymes in the body [34]. Data on the metabolism of PEGylated proteins in human subjects are not available, although there is clear evidence that degradation occurs in animals, leading to the release of PEG.…”

Disposition of Basal Insulin Peglispro Compared with 20-kDa Polyethylene Glycol in Rats Following a Single Intravenous or Subcutaneous Dose

“…Radioactivity was still being eliminated in urine and feces at the end of the 2 weeks, which was not unexpected given the long elimination t 1/2 for 14 C. The recovery of total radioactivity in other studies with PEGylated molecules has been reported to be similarly low: 38%-80% for 46-kDa PEGylated [ 3 H]neuromedin-U receptor agonist in rat and dog (Zou et al, 2013) and 53%-67% for 2 Â 20-kDa PEGylated [ 14 C] peginesatide in rats and monkeys (Woodburn et al, 2012(Woodburn et al, , 2013. In a study with nonradiolabeled 2 Â 20-kDa PEG insulin, Elliott et al (2012) reported recovery of 35%-47% of the PEG dose in urine after 28 days. These studies and ours demonstrate the difficulty of obtaining complete recovery of the dose when PEGylated molecules are administered.…”

“…The fate of therapeutic proteins is generally thought to be via catabolism to small peptides and amino acids, but the biologic fate of PEGylated proteins has not been well studied because of the difficulties in quantifying PEG. The fate of a 40-kDa PEG attached to insulin has been determined by using immunoblotting with antibodies to PEG combined with NMR spectroscopy (Elliott et al, 2012), and the tissue uptake of 40-kDa branched PEG on Adnectin was determined by using a 14 C-labeled cross-linker on 40-kDa branched PEGylated Adnectin (Wang et al, 2012a). The disposition of […”

“…NMR spectroscopy has been used to analyze the biologic fate of a PEGylated protein; however, a full mass balance could not be established with NMR spectroscopy, and these experiments could not tease apart the fates of the intact molecule and metabolites with PEG attached. There are reports of studies using immunoblotting with antibodies to PEG combined with NMR spectroscopy to assess the tissue uptake of a 40-kDa PEG moiety attached to insulin (Elliott et al, 2012 (Wang et al, 2012a), 2 Â 20-kDa PEGylated […”