Monitoring the Glycosylation Status of Proteins Using Raman Spectroscopy

Abstract

Protein-based biopharmaceuticals are becoming increasingly widely used as therapeutic agents, and the characterization of these biopharmaceuticals poses a significant analytical challenge. In particular, monitoring posttranslational modifications (PTMs), such as glycosylation, is an important aspect of this characterization because these glycans can strongly affect the stability, immunogenicity, and pharmacokinetics of these biotherapeutic drugs. Raman spectroscopy is a powerful tool, with many emerging applications in the bioprocessing arena. Although the technique has a relatively rich history in protein science, only recently has Raman spectroscopy been investigated for assessing posttranslational modifications, including phosphorylation, acetylation, trimethylation, and ubiquitination. In this investigation, we develop for the first time Raman spectroscopy combined with multivariate data analyses, including principal components analysis and partial least-squares regression, for the determination of the glycosylation status of proteins and quantifying the relative concentrations of the native ribonuclease (RNase) A protein and RNase B glycoprotein within mixtures.

Raman Spectroscopy Provides a Powerful Diagnostic Tool for Accurate Determination of Albumin Glycation

“…The next step in translating this promising technology is to assess its predictive diagnostic value in multi-component mixtures, especially in serum samples. Additionally, in combination with recent studies of Raman-based characterization of protein glycosylation status [76], our investigations should advance the use of Raman and other spectroscopic modalities (such as fluorescence, FTIR and 2D-IR absorption spectroscopy [77]) for understanding the detailed structure and dynamics of albumin transformation caused by different analytes of interest, such as glucose and heavy metal ions [78].…”

Raman Spectroscopy-Based Sensitive and Specific Detection of Glycated Hemoglobin

“…This is particularly significant given the substantive implications of protein glycosylation for the stability, pharmacokinetics and immunogenicity of glycoprotein-based biopharmaceuticals. 46, 47 …”

Monitoring Preparation of Derivative Protein Crystals via Raman Microscopy

“…In these crystals, a quantitative evaluation of the relative amount of SeMet replacement was also achieved by comparative analysis. In principle, Raman microspectra could be also used to reveal post translational modifications, such as phosphorylation, acetylation, trimethylation, ubiquitination and glycosilation, as already done by comparing spectra of native and derivative proteins in solution (Sundararajan, et al 2006;Brewster et al, 2011).…”