Detection of glycosylation and iron-binding protein modifications using Raman spectroscopy.

In this study we demonstrate the use of Raman spectroscopy to determine protein modifications as a result of glycosylation and iron binding. Most proteins undergo some modifications after translation which can directly affect protein function. Identifying these modifications is particularly important in the production of biotherapeutic agents as they can affect stability, immunogenicity and pharmacokinetics. However, post-translational modifications can often be difficult to detect with regard to the subtle structural changes they induce in proteins. From their Raman spectra apo-and holo-forms of iron-binding proteins, transferrin and ferritin, could be readily distinguished and variations in spectral features as a result of structural changes could also be determined. In particular, differences in solvent exposure of aromatic amino acids residues could be identified between the open and closed forms of the iron-binding proteins. Protein modifications as a result of glycosylation can be even more difficult to identify. Through the application of the chemometric techniques of principal component analysis and partial least squares regression variations in Raman spectral features as a result of glycosylation induced structural modifications could be identified. These were then used to distinguish between glycosylated and non-glycosylated transferrin and to measure the relative concentrations of the glycoprotein within a mixture of the native non-glycosylated protein.

Detection of Protein Glycosylation Using Tip-Enhanced Raman Scattering.

The correct glycosylation of biopharmaceutical glycoproteins and their formulations is essential for them to have the desired therapeutic effect on the patient. It has recently been shown that Raman spectroscopy can be used to quantify the proportion of glycosylated protein from mixtures of native and glycosylated forms of bovine pancreatic ribonuclease (RNase). Here we show the first steps toward not only the detection of glycosylation status but the characterization of glycans themselves from just a few protein molecules at a time using tip-enhanced Raman scattering (TERS). While this technique generates complex data that are very dependent on the protein orientation, with the careful development of combined data preprocessing, univariate and multivariate analysis techniques, we have shown that we can distinguish between the native and glycosylated forms of RNase. Many glycoproteins contain populations of subtly different glycoforms; therefore, with stricter orientation control, we believe this has the potential to lead to further glycan characterization using TERS, which would have use in biopharmaceutical synthesis and formulation research.

The challenge of applying Raman spectroscopy to monitor recombinant antibody production.

UV resonance Raman (UVRR) spectroscopy combined with chemometric techniques was investigated as a physiochemical tool for monitoring secreted recombinant antibody production in cultures of Chinese hamster ovary (CHO) cells. Due to the enhanced selectivity of the UVRR, spectral variations arising from protein, small molecule substrates, and nucleic acid medium components could be measured simultaneously and we have successfully determined antibody titre. Medium samples were taken during culture of three CHO cell lines: two antibody-producing cell lines and a non-producing cell line, and analysed by UVRR spectroscopy using an excitation laser of 244 nm. Principal component analysis (PCA) was applied to the spectral sets and showed a linear trend over time for the antibody-producing cell lines that was not observed in the non-producing cell line. Partial least squares regression (PLSR) was used to predict antibody titres, glucose utilization and lactate accumulation, and compared very favourably with gold standard data acquired with the much slower techniques of ELISA and liquid chromatography. Further analysis of the UVRR spectral sets using two-dimensional correlation moving windows also revealed that spectral variations due to protein and nucleic acid concentrations in the medium during cell culture varied between each of the three cell lines investigated.

Monitoring guanidinium-induced structural changes in ribonuclease proteins using Raman spectroscopy and 2D correlation analysis.

Assessing the stability of proteins by comparing their unfolding profiles is a very important characterization and quality control step for any biopharmaceutical, and this is usually measured by fluorescence spectroscopy. In this paper we propose Raman spectroscopy as a rapid, noninvasive alternative analytical method and we shall show this has enhanced sensitivity and can therefore reveal very subtle protein conformational changes that are not observed with fluorescence measurements. Raman spectroscopy is a powerful nondestructive method that has a strong history of applications in protein characterization. In this work we describe how Raman microscopy can be used as a fast and reliable method of tracking protein unfolding in the presence of a chemical denaturant. We have compared Raman spectroscopic data to the equivalent samples analyzed using fluorescence spectroscopy in order to validate the Raman approach. Calculations from both Raman and fluorescence unfolding curves of [D]50 values and Gibbs free energy correlate well with each other and more importantly agree with the values found in the literature for these proteins. In addition, 2D correlation analysis has been performed on both Raman and fluorescence data sets in order to allow further comparisons of the unfolding behavior indicated by each method. As many biopharmaceuticals are glycosylated in order to be functional, we compare the unfolding profiles of a protein (RNase A) and a glycoprotein (RNase B) as measured by Raman spectroscopy and discuss the implications that glycosylation has on the stability of the protein.

Monitoring of the interconversion of gamma-butyrolactone (GBL) to gamma hydroxybutyric acid (GHB) by Raman spectroscopy.

Gamma-hydroxybutyric acid (GHB) is a drug-of-abuse that has recently become associated with drug-facilitated sexual assault, known as date rape. For this reason the drug is commonly found 'spiked' in alcoholic beverages. When GHB is in solution it may undergo conversion into the corresponding lactone, Gamma-butyrolactone (GBL). Studies have been carried out to determine the detection limits of GHB and GBL in various solutions by Raman spectroscopy and to monitor the interconversion of GHB and GBL in solution with different pH conditions and temperature. In this study, a portable Raman spectrometer was used to study the interconversion of GHB and GBL in water and ethanol solutions as a function of pH, time, and temperature. The aim of this was to determine the optimum pH range for conversion in order to relate this to the pH ranges that the drug is likely to be subjected to, first in spiked beverages and secondly after ingestion in the digestive system. The aim was also to identify a timescale for this conversion in relation to possible scenarios, for example if GHB takes a number of hours to convert to GBL, it is likely for the beverage to be ingested before esterification can take place. GHB and GBL were then spiked into a selection of beverages of known pH in order to study the stability of GHB and GBL in real systems.

Fingerprinting food: current technologies for the detection of food adulteration and contamination.

Major food adulteration and contamination events seem to occur with some regularity, such as the widely publicised adulteration of milk products with melamine and the recent microbial contamination of vegetables across Europe for example. With globalisation and rapid distribution systems, these can have international impacts with far-reaching and sometimes lethal consequences. These events, though potentially global in the modern era, are in fact far from contemporary, and deliberate adulteration of food products is probably as old as the food processing and production systems themselves. This review first introduces some background into these practices, both historically and contemporary, before introducing a range of the technologies currently available for the detection of food adulteration and contamination. These methods include the vibrational spectroscopies: near-infrared, mid-infrared, Raman; NMR spectroscopy, as well as a range of mass spectrometry (MS) techniques, amongst others. This subject area is particularly relevant at this time, as it not only concerns the continuous engagement with food adulterers, but also more recent issues such as food security, bioterrorism and climate change. It is hoped that this introductory overview acts as a springboard for researchers in science, technology, engineering, and industry, in this era of systems-level thinking and interdisciplinary approaches to new and contemporary problems.

Monitoring the glycosylation status of proteins using Raman spectroscopy.

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.

Identification of the date-rape drug GHB and its precursor GBL by Raman spectroscopy.

Gamma hydroxybutyric acid (GHB), also known as 'liquid ecstasy', has recently become associated with drug-facilitated sexual assaults, known colloquially as 'date rape', due to the ability of the drug to cause loss of consciousness. The drug is commonly found 'spiked' into alcoholic beverages, as alcohol increases its sedative effects. Gamma hydroxybutyric acid and the corresponding lactone gamma-butyrolactone (GBL) will reach an equilibrium in solution which favours the lactone in basic conditions and GHB in acidic conditions (less than pH 4). Therefore, we have studied both GHB and GBL, as a mildly acidic beverage 'spiked' with GHB will contain both GHB and GBL. We report the analysis of GHB as a sodium salt and GBL, its precursor, using bench-top and portable Raman spectroscopy. It has been demonstrated that we are able to detect GHB and GBL in a variety of containers including colourless and amber glass vials, plastic vials and polythene bags. We have also demonstrated the ability to detect both GBL and GHB in a range of liquid matrices simulating 'spiked' beverages.