Quantitative analysis of polyethylene glycol (PEG) and PEGylated proteins in animal tissues by LC-MS/MS coupled with in-source CID.

The covalent conjugation of polyethylene glycol (PEG, typical MW > 10k) to therapeutic peptides and proteins is a well-established approach to improve their pharmacokinetic properties and diminish the potential for immunogenicity. Even though PEG is generally considered biologically inert and safe in animals and humans, the slow clearance of large PEGs raises concerns about potential adverse effects resulting from PEG accumulation in tissues following chronic administration, particularly in the central nervous system. The key information relevant to the issue is the disposition and fate of the PEG moiety after repeated dosing with PEGylated proteins. Here, we report a novel quantitative method utilizing LC-MS/MS coupled with in-source CID that is highly selective and sensitive to PEG-related materials. Both (40K)PEG and a tool PEGylated protein (ATI-1072) underwent dissociation in the ionization source of mass spectrometer to generate a series of PEG-specific ions, which were subjected to further dissociation through conventional CID. To demonstrate the potential application of the method to assess PEG biodistribution following PEGylated protein administration, a single dose study of ATI-1072 was conducted in rats. Plasma and various tissues were collected, and the concentrations of both (40K)PEG and ATI-1072 were determined using the LC-MS/MS method. The presence of (40k)PEG in plasma and tissue homogenates suggests the degradation of PEGylated proteins after dose administration to rats, given that free PEG was absent in the dosing solution. The method enables further studies for a thorough characterization of disposition and fate of PEGylated proteins.

Utility of quantitative whole-body autoradiography (QWBA) and imaging mass spectrometry (IMS) by matrix-assisted laser desorption/ionization (MALDI) in the assessment of ocular distribution of drugs.

INTRODUCTION: Assessment of drug candidate properties and potential liabilities can greatly benefit from issue driven studies that are designed to address specific toxicological effects such as ocular phototoxicity. If a compound absorbs light in the wavelength range of 290-700 nm (UV-A, UV-B, and visible light) and generates a positive response in a standard in vitro neutral red uptake phototoxicity assay in Balb/c 3T3 mouse fibroblasts, a single-dose in vivo study may be conducted to assess the potential for drug-induced phototoxicity in the eyes and skin of pigmented Long-Evans rats. Critical to ocular phototoxicity assessment is the hypothesis that the drug or drug-related material must be present in the affected substructures such as the uveal tract, retina, lens, or cornea. For compounds that induce a positive ocular response in the in vivo phototoxicity assay, data on distribution patterns to substructures of the eye can inform decisions regarding the nature of the ocular findings and possibly influence compound advancement. METHODS: Quantitative whole-body autoradiography (QWBA) and imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization (MALDI) on an ion trap mass spectrometer employing higher order mass spectrometric scanning functions were utilized for localization of dosed drug or metabolites in eye substructures. RESULTS: In investigative studies designed to simulate an in vivo phototoxicity study, rats were administered radio-labeled test article for QWBA analysis and un-labeled test article for IMS analysis. Autoradiograms from the QWBA study indicated that the radio-labeled analyte(s) preferentially distributed to the uveal tract and not the cornea. However, QWBA did not provide information on the nature of the detected analyte(s); i.e. intact parent drug versus potential metabolites or degradants. Multistage MS experiments performed directly on tissue sections demonstrated semi-quantitative localization in the uveal tract and unequivocal identification of the analyte as the dosed parent drug; no potential metabolites were detected. DISCUSSION: Image analysis by QWBA and IMS by MALDI proved complementary in the localization and identification of small molecule drug distribution within the eye.