Potential of Time Domain Nuclear Magnetic Resonance as a Noninvasive Method for Detection and Quantification of Protein Glycation in Biopharmaceuticals

Biopharmaceuticals are an essential and growing part of modern medicine. Given their complex structure, they are prone to chemical and physical instabilities, including glycation, a nonenzymatic chemical reaction between the free amino groups of proteins and reducing saccharides. Glycation reduces the drug's efficacy and can produce harmful compounds in the body. Current detection methods, such as liquid chromatography-mass spectrometry, affinity chromatography, and chemical reaction methods, including the <i>O</i>-phthalaldehyde (OPA) method, are invasive, labor-intensive, and costly. This study presents time domain nuclear magnetic resonance (TD-NMR) as a noninvasive, low-cost, and user-friendly method for the detection and quantification of glycation in biopharmaceuticals. Bovine serum albumin and glucose were employed as a model system and were placed under various temperatures and durations to induce glycation. Several TD-NMR techniques, including one-dimensional longitudinal (<i>T</i>₁) and transverse (<i>T</i>₂) relaxation times, as well as 2-D <i>T</i>₁<i>T</i>₂ maps, were applied to the glycated samples, providing a better understanding of the hydration behavior and water mobility during glycation. Comparing these results to the OPA method had shown that both <i>T</i>₁ and <i>T</i>₂ values change proportionally with glycation, indicating alterations in molecular mobility and hydration. <i>T</i>₁/<i>T</i>₂ map values were highly correlated with the OPA method (<i>p</i> < 0.05, RMSE ≤ 0.12, MAE ≤ 0.10), supporting the applicability of TD-NMR as a quantitative glycation measurement tool. Additional analyses, such as protein quantification, secondary structure evaluation, and assessment of early and late glycation products, further confirmed the relevance of TD-NMR in monitoring structural and chemical changes during glycation.