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Quantitative Detection of PEGylated Biomacromolecules in Biological Fluids by NMR.

Alvares, Rohan D A; Hasabnis, Advait; Prosser, R Scott; Macdonald, Peter M.
Anal Chem; 88(7): 3730-8, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-26927487
The accumulation, biodistribution, and clearance profiles of therapeutic agents are key factors relevant to their efficacy. Determining these properties constitutes an ongoing experimental challenge. Many such therapeutics, including small molecules, peptides, proteins, tissue scaffolds, and drug delivery vehicles, are conjugated to poly(ethylene glycol) (PEG) as this improves their bioavailability and in vivo stability. We demonstrate here that (1)H NMR spectroscopy can be used to quantify PEGylated species in complex biological fluids directly, rapidly, and with minimal sample preparation. PEG bears a large number of spectroscopically equivalent protons exhibiting a narrow NMR line width while resonating at a (1)H NMR frequency distinct from most other biochemical signals. We demonstrate that PEG provides a robust signal allowing detection of concentrations as low as 10 µg/mL in blood. This PEG detection limit is lowered by another order of magnitude when background proton signals are minimized using (13)C-enriched PEG in combination with a double quantum filter to remove (1)H signals from non-(13)C-labeled species. Quantitative detection of PEG via these methods is shown in pig blood and goat serum as examples of complex biological fluids. More practically, we quantify the blood clearance of (13)C-PEG and PEGylated-BSA (bovine serum albumin) following their intravenous injection in live rats. Given the relative insensitivity of line width to PEG size, we anticipate that the biodistribution and clearance profiles of virtually any PEGylated biomacromolecule from biological fluid samples can be routinely measured by (1)H NMR without any filtering or treatment steps.