Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies

Davies, Heather; Singh, Prabha; Deckert‐Gaudig, Tanja; Deckert, Volker; Rousseau, Karine; Ridley, Caroline; Dowd, Sarah E; Doig, Andrew J.; Pudney, Paul D. A.; Thornton, David J.; Blanch, Ewan W.

doi:10.1021/acs.analchem.6b03095

Cited by 41 publications

(34 citation statements)

References 49 publications

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“…This observation is consistent with the fact that the central mucin polymers are inherently randomly coiled due to the heavy glycosylation and the presence of negatively charged sugars that prevent the ordering of the mucin domains (Figure B) . A considerable presence of the β‐sheets has also been found in certain mucins, such as MUC5B, which is deemed critical for the globular domain specific functions, such as the intermolecular interactions, cross‐linking, and polymerization . The presence of β‐sheet amide I peaks confirmed the existence of these domains in MUC2 that constituted the porcine jejunal mucus.…”

Section: Resultssupporting

confidence: 79%

Shedding Light on the Trehalose‐Enabled Mucopermeation of Nanoparticles with Label‐Free Raman Spectroscopy

Siddhanta

Bhattacharjee

Harrison

et al. 2019

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Nanoparticle‐based drug delivery systems have attracted significant interest owing to their promise as tunable platforms that offer improved intracellular release of cargo therapeutics. However, significant challenges remain in maintaining the physiological stability of the mucosal matrix due to the nanoparticle‐induced reduction in the matrix diffusivity and promotion of mucin aggregation. Such aggregation also adversely impacts the permeability of the nanoparticles, and thus, diminishes the efficacy of nanoparticle‐based formulations. Here, an entirely complementary approach is proposed to the existing nanoparticle functionalization methods to address these challenges by using trehalose, a naturally occurring disaccharide that offers exceptional protein stabilization. Plasmon‐enhanced Raman spectroscopy and far‐red fluorescence emission of the plasmonic silver nanoparticulate clusters are harnessed to create a unique dual‐functional, aggregating, and imaging agent that obviates the need of an additional reporter to investigate mucus–nanoparticle interactions. These spectroscopy‐based density mapping tools uncover the mechanism of mucus–nanoparticle interactions and establish the protective role of trehalose microenvironment in minimizing the nanoparticle aggregation. Thus, in contrast to the prevailing belief, these results demonstrate that nonfunctionalized nanoparticles may rapidly penetrate through mucus barriers, and by leveraging the bioprotectant attributes of trehalose, an in vivo milieu for efficient mucosal drug delivery can be generated.

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Section: Resultssupporting

confidence: 79%

Shedding Light on the Trehalose‐Enabled Mucopermeation of Nanoparticles with Label‐Free Raman Spectroscopy

Siddhanta

Bhattacharjee

Harrison

et al. 2019

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“…As a response to T cell stimulation, major transcriptional activity begins within the nucleus (36), an effect that manifests in the difference Raman spectra of T cells upon LPS stimulation for 4 d. Accordingly, relatively strong changes are detected in the Raman band at ;795 cm 21 , arising from the nucleus moiety (18,33), which can be assigned to phosphate backbone vibration (15,35,37). At https://doi.org/10.4049/immunohorizons.1800059 (Fig.…”

Section: Cd4 + and Cd8 + T Cell Analysismentioning

confidence: 99%

Raman Spectroscopy Follows Time-Dependent Changes in T Lymphocytes Isolated from Spleen of Endotoxemic Mice

et al. 2019

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T lymphocytes (T cells) are highly specialized members of the adaptive immune system and hold the key to the understanding the hosts' response toward invading pathogen or pathogen-associated molecular patterns such as LPS. In this study, noninvasive Raman spectroscopy is presented as a label-free method to follow LPS-induced changes in splenic T cells during acute and postacute inflammatory phases (1, 4, 10, and 30 d) with a special focus on CD4 + and CD8 + T cells of endotoxemic C57BL/6 mice. Raman spectral analysis reveals highest chemical differences between CD4 + and CD8 + T cells originating from the control and LPS-treated mice during acute inflammation, and the differences are visible up to 10 d after the LPS insult. In the postacute phase, CD4 + and CD8 + T cells from treated and untreated mice could not be differentiated anymore, suggesting that T cells largely regained their original status. In sum, the biological information obtained from Raman spectra agrees with immunological readouts demonstrating that Raman spectroscopy is a well-suited, label-free method for following splenic T cell activation in systemic inflammation from acute to postacute phases. The method can also be applied to directly study tissue sections as is demonstrated for spleen tissue one day after LPS insult. ImmunoHorizons, 2019, 3: 45-60.

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“…182 Finally, the combination of TERS, ROA, and CD spectroscopies was used successfully to study some aspects of the structure and glycosylation of mucin proteins. 183 Unfortunately, the implementation of a Raman spectroscopy-based method capable of displacing MS-based methods for MAb glycosylation analysis is likely to prove impossible for the time-being. This is because industry has a need for very comprehensive glycan and glycoform characterization to meet regulatory requirements and the sheer complexity and variability of glycosylation in terms of sites, branching, micro-heterogeneity, and macroheterogeneity, produce very complex Raman spectra that are intrinsically difficult to analyze.…”

Section: Analysis Of Biopharmaceutical Productsmentioning

confidence: 99%

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

2017

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The production of active pharmaceutical ingredients (APIs) is currently undergoing its biggest transformation in a century. The changes are based on the rapid and dramatic introduction of protein-and macromolecule-based drugs (collectively known as biopharmaceuticals) and can be traced back to the huge investment in biomedical science (in particular in genomics and proteomics) that has been ongoing since the 1970s. Biopharmaceuticals (or biologics) are manufactured using biological-expression systems (such as mammalian, bacterial, insect cells, etc.) and have spawned a large (>E35 billion sales annually in Europe) and growing biopharmaceutical industry (BioPharma). The structural and chemical complexity of biologics, combined with the intricacy of cell-based manufacturing, imposes a huge analytical burden to correctly characterize and quantify both processes (upstream) and products (downstream). In small molecule manufacturing, advances in analytical and computational methods have been extensively exploited to generate process analytical technologies (PAT) that are now used for routine process control, leading to more efficient processes and safer medicines. In the analytical domain, biologic manufacturing is considerably behind and there is both a huge scope and need to produce relevant PAT tools with which to better control processes, and better characterize product macromolecules. Raman spectroscopy, a vibrational spectroscopy with a number of useful properties (nondestructive, non-contact, robustness) has significant potential advantages in BioPharma. Key among them are intrinsically high molecular specificity, the ability to measure in water, the requirement for minimal (or no) sample pre-treatment, the flexibility of sampling configurations, and suitability for automation. Here, we review and discuss a representative selection of the more important Raman applications in BioPharma (with particular emphasis on mammalian cell culture). The review shows that the properties of Raman have been successfully exploited to deliver unique and useful analytical solutions, particularly for online process monitoring. However, it also shows that its inherent susceptibility to fluorescence interference and the weakness of the Raman effect mean that it can never be a panacea. In particular, Raman-based methods are intrinsically limited by the chemical complexity and wide analyte-concentration-profiles of cell culture media/bioprocessing broths which limit their use for quantitative analysis. Nevertheless, with appropriate foreknowledge of these limitations and good experimental design, robust analytical methods can be produced. In addition, new technological developments such as time-resolved detectors, advanced lasers, and plasmonics offer potential of new Raman-based methods to resolve existing limitations and/or provide new analytical insights.

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Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies

Cited by 41 publications

References 49 publications

Shedding Light on the Trehalose‐Enabled Mucopermeation of Nanoparticles with Label‐Free Raman Spectroscopy

Shedding Light on the Trehalose‐Enabled Mucopermeation of Nanoparticles with Label‐Free Raman Spectroscopy

Raman Spectroscopy Follows Time-Dependent Changes in T Lymphocytes Isolated from Spleen of Endotoxemic Mice

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

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