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Abstract
Protein post-translational modifications (PTMs), including glycation, glycosylation, and pyroglutamation could impact protein function and stability. Liquid chromatography coupled with mass spectrometry (LC-MS) is a vital tool for PTM characterization, with hydrophilic interaction liquid chromatography (HILIC) emerging as the preferred method for characterization of hydrophilic modifications. This research focuses on improving the characterization of PTMs such as glycation and pyroglutamation by predicting the retention times of modified peptides in the HILIC-MS mode. Glycation has been linked to health conditions including diabetes, cataract and Alzheimer's disease. This modification could be introduced during the manufacturing of therapeutic monoclonal antibodies (mAbs), necessitating its accurate characterization. Thus, an analytical workflow was developed to predict and characterize glycation sites in mAbs with the aim to enhance safety and efficacy of drug products. In addition, retention prediction of peptides containing pyroglutamated N-terminus glutamic acid was performed. Pyroglutamate formation is a PTM that has been associated with several neurodegenerative disorders. Such a predictive approach is expected to serve as an additional layer of validation in PTM characterizations, ultimately leading to faster analysis times. Further, absolute and relative quantitation of glycation modification using heavy isotope-labeled proteins containing 13C and 15N lysine and arginine has been developed. The proposed method was observed to have higher accuracy and precision compared to the typically established method for glycation quantitation. This quantitative approach was applied to IgGs such as Adalimumab (IgG1), Natalizumab (IgG4), and 'Frankenmab' (custom IgG2).The proposed research also evaluates various LC-MS sample preparation techniques that are employed for N-glycosylation characterizations. The choice of LC-MS sample preparation is crucial to avoid inaccuracies in determining the absolute and relative abundances of N-glycans.
Lastly, this work provides valuable insights into the operations of shared resource laboratories (SRLs) that accommodate analytical instruments. A survey was conducted to better understand instrument usage, maintenance, associated costs, and funding sources in SRLs specializing in flow cytometry.