UV Resonance Raman Spectroscopy Using a New cw Laser Source: Convenience and Experimental Simplicity

Asher, Sanford A.; Bormett, Richard W.; Chen, X. G.; Lemmon, Donald H.; Cho, Namjun; Peterson, Pete; Arrigoni, Marco; Spinelli, Luis; Cannon, J.

doi:10.1366/0003702934067225

Cited by 111 publications

(80 citation statements)

References 32 publications

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“…the laser excitation source can contribute >50% of system costs, compared to $10% for CRS). 94 These demands have prevented its widespread adoption outside of academia, however, the component technologies are advancing all the time and the rapid pace of photonics developments in the past decade suggests that the relevant technological issues can be solved (particularly if instrument manufacturers can leverage technologies being developed for semiconductor manufacture using extreme UV).…”

Section: Raman Spectroscopymentioning

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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Section: Raman Spectroscopymentioning

confidence: 99%

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

2017

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“…Using 229 nm excitation (10), the tryptophan and tyrosine Raman bands are selectively enhanced (11,12), enabling UVRR spectroscopy to provide structural and environmental information concerning the side chains of these aromatic residues. Here, we report UVRR studies of the native structures of Im7* and Im9*, together with three variants of Im7* [termed L53AI54A, H3G6, and YY (inset of Figure 1A)], that trap a partially folded state of Im7* at equilibrium by preventing the binding of helix III to the developing structure (13).…”

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confidence: 99%

Ultraviolet Resonance Raman Studies Reveal the Environment of Tryptophan and Tyrosine Residues in the Native and Partially Folded States of the E Colicin-Binding Immunity Protein Im7

et al. 2005

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Understanding the nature of partially folded proteins is a challenging task that is best accomplished when several techniques are applied in combination. Here we present ultraviolet resonance Raman (UVRR) spectroscopy studies of the E colicin-binding immunity proteins, Im7* and Im9*, together with a series of variants of Im7* that are designed to trap a partially folded state at equilibrium. We show that the environments of the tryptophan and tyrosine residues in native wild-type Im7* and Im9* are indistinguishable, in contrast with models for their structures based on X-ray and NMR methods. In addition, we show that there is a general increase in the hydrophobicity in the environment of Trp75 in all of the variants compared with wild-type Im7*. These data suggest that a significant rearrangement of the tryptophan pocket occurs in the variants, which, together with an overall decrease in solvent accessibility of Trp75 as judged by time-resolved fluorescence lifetime measurements and fluorescence quenching experiments, rationalize the unusual fluorescence properties of the variants reported previously. The data highlight the power of UVRR in analyzing the structural properties of different conformational states of the same protein and reveal new information about the structural rearrangements occurring during Im7* folding, not possible using other spectroscopic methods alone. Finally, we describe a previously unreported dependence of the tryptophan Fermi doublet on excitation wavelength in the ultraviolet region revealed by these protein spectra. We corroborated this observation using tryptophan-containing model compounds and conclude that the conventional interpretation of this UVRR feature at these wavelengths is unreliable.The biological role of the E colicin-binding immunity proteins Im2, Im7, Im8, and Im9 is to protect the host cell from the toxic endonuclease activity of a coexpressed cognate colicin, by binding to an exosite in the DNase domain, inhibiting access of the substrate DNA to the active site of the colicin by steric hindrance and electrostatic repulsion (1, 2). The small size of the immunity proteins (85-87 amino acid residues), their high level of sequence identity (approximately 60%), the single conserved tryptophan, and their lack of disulfide bonds, prosthetic groups, and cis-prolines in the native state make the immunity protein family ideal for protein folding studies. Despite the high degree of similarity between Im7 and Im9 in both sequence and structure, these proteins fold by mechanisms with different levels of kinetic complexity at neutral pH, suggesting that local sequence variations, rather than topology or global stability, are responsible for the observed folding kinetics of these proteins (3-7). Im7* 1 and Im9* (hexahistidinetagged analogues of Im7 and Im9) contain four helices ( Figure 1A), each of which displays polar side chains on the surface of the protein, while the majority of hydrophobic side chains are buried, forming a well-packed hydrophobic core. Im7* and Im...

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“…Thirteen discrete lines have been shown to have lasing powers in excess of 20 m W . 23 This cw capability in the UV will have tremendous impact on the use of resonance Raman spectroscopy for structural studies. Previously, pulsed lasers had to be used to obtain UV output.…”

Section: Fig 3 Optical Layout Of An On-axis Low-f-number Holographmentioning

confidence: 99%

A New Generation of Raman Instrumentation

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1994

Appl Spectrosc

101

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UV Resonance Raman Spectroscopy Using a New cw Laser Source: Convenience and Experimental Simplicity

Cited by 111 publications

References 32 publications

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

Ultraviolet Resonance Raman Studies Reveal the Environment of Tryptophan and Tyrosine Residues in the Native and Partially Folded States of the E Colicin-Binding Immunity Protein Im7

A New Generation of Raman Instrumentation

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