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

## TL;DR

This paper explores using time domain nuclear magnetic resonance as a noninvasive and cost-effective way to detect and measure protein glycation in biopharmaceuticals.

## Contribution

The study introduces TD-NMR as a novel, noninvasive method for quantifying glycation in biopharmaceuticals.

## Key findings

- TD-NMR relaxation times (T1 and T2) correlate with glycation levels, showing changes in molecular mobility and hydration.
- T1/T2 map values from TD-NMR are highly correlated with the OPA method (p < 0.05, RMSE ≤ 0.12, MAE ≤ 0.10).
- TD-NMR can monitor structural and chemical changes during glycation, including early and late glycation products.

## Abstract

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 O-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 (T
1) and transverse (T
2) relaxation times, as well as 2-D T
1
T
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 T
1 and T
2 values change proportionally
with glycation, indicating alterations in molecular mobility and hydration. T
1/T
2 map values
were highly correlated with the OPA method (p <
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.