Deciphering the In Vivo Fate of PEGylated rhGH via a Molecular Ruler-Based MS Strategy

Abstract A growing number of long-acting protein therapeutics rely on high-molecular-weight (HMW) polyethylene glycol (PEG) conjugation to extend circulation time, yet the in vivo fate of PEG-derived species remains a critical blind spot for drug safety and regulatory evaluation. A key analytical challenge is that intact conjugates and PEG-derived species cannot be resolved by intact-protein or total-PEG measurements alone. Here we report a molecular ruler-based mass spectrometry strategy that integrates MSALL acquisition, PEG standard calibration anchors spanning broad molecular-weight ranges, and a self-built high-resolution mass spectral (HRMS) library to convert broad, overlapping PEG signal envelopes into temporally traceable, molecular-weight-referenced analytical targets. Applied to 40 kDa PEG-modified recombinant human growth hormone (PEG-rhGH) in rats, the workflow quantitatively traced the intact conjugate (plasma half-life t1/2 = 26.83 h) and concurrently resolved tissue-associated PEG 40 kDa and a PEG-related metabolite series. Intact PEG-rhGH remained below the validated tissue LLOQ, whereas PEG-derived components accumulated preferentially in the liver (peak at 48 h), with total urinary and fecal recovery of the quantitatively monitored intact PEG-rhGH and PEG 40 kDa below 10% by 144 hindicating profoundly delayed elimination. HRMS interrogation revealed low-molecular-weight PEG oligomers (PEG7−PEG21) together with carboxylic-acid- and aldehyde-terminated species in liver, consistent with in vivo chain shortening and end-group transformation. By linking intact-conjugate exposure, tissue accumulation, metabolite profiling, and excretion within a single transferable framework, this strategy provides a measurement-science tool for the systematic evaluation of HMW PEGylated protein therapeutics and other pharmaceutical polymer systems.