PEGylation is the process of covalent attachment of polyethylene glycol (PEG) polymerchains to a peptide or therapeutic protein with the goal of improving the pharmacokinetic behavior of protein by, reducing glomerular filtration and increased resistance to clearance via proteolytic enzymes. While PEGylation of peptides/proteins generally improves the PK and PK/PD of the therapeutic drugs, the disposition of PEGylated proteins in animals has caused some toxicological concern as multiple PEGylated proteins have been shown to lead to tissue vaculation presumably due to PEG accumulation.The tissue accumulation has not been fully characterized due to the heterogeneous nature of the PEG molecules themselves and the PEGylated protein and the limitation of current technologies for PEG detection and quantitation. A novel LC/MS/MS based methodology was developed to directly quantify the PEG and PEGylated protein in biological samples such as serum, or homogenates prepared from liver, kidney, and brain after dosing of PEG or a PEGylated protein (PEGylated version of an antibody fragment). This method relies on the in-source fragmentation of PEG and the further CID fragmentation in Q2 of the most abundant MS fragment resulting from in-source decay. A liquid-liquid extraction method was used to extract PEGylated proteins and PEG from various biological samples. The recovery of this extraction process was characterized by the use a radiolabeled PEG conjugate. Sufficient linearity and dynamic range (0.5-250 �g/mL) were achieved for the quantitation of PEG and PEG-proteins. Intraday and interday accuracy and precision from QC samples were within � 20%. This method was successfully applied to in vivo samples such as serum, liver, kidney, and brain analysis after administration of a PEGylated antibody fragment.

Direct quantification of PEG and PEGylated protein conjugate in biological samples using in-source CID and LC-MS/MS analysis

248th National Meeting (2014)

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