Infrared absorbance spectroscopy of aqueous proteins : comparison of transmission and ATR data collection and analysis for secondary structure fitting

Attenuated total reflectance (ATR) infrared absorbance spectroscopy of proteins in aqueous solution is much easier to perform than transmission spectroscopy, where short path‐length cells need to be assembled reproducibly. However, the shape of the resulting ATR infrared spectrum varies with the refractive index of the sample and the instrument configuration. Refractive index in turn depends on the absorbance of the sample. In this work, it is shown that a room temperature triglycine sulfate detector and a ZnSe ATR unit can be used to collect reproducible spectra of proteins. A simple method for transforming the protein ATR spectrum into the shape of the transmission spectrum is also given, which proceeds by approximating a Kramers‐Krönig–determined refractive index of water as a sum of four linear components across the amide I and II regions. The light intensity at the crystal surface (with 45° incidence) and its rate of decay away from the surface is determined as a function of the wave number–dependent refractive index as well as the decay of the evanescent wave from the surface. The result is a single correction factor at each wave number. The spectra were normalized to a maximum of 1 between 1600 cm−1 and 1700 cm−1 and a self‐organizing map secondary structure fitting algorithm, SOMSpec, applied using the BioTools reference set. The resulting secondary structure estimates are encouraging for the future of ATR spectroscopy for biopharmaceutical characterization and quality control applications

SOMSpec as a general purpose validated self-organising map tool for rapid protein secondary structure prediction from infrared absorbance data

A protein’s structure is the key to its function. As protein structure can vary with environment, it is important to be able to determine it over a wide range of concentrations, temperatures, formulation vehicles, and states. Robust reproducible validated methods are required for applications including batch-batch comparisons of biopharmaceutical products. Circular dichroism is widely used for this purpose, but an alternative is required for concentrations above 10 mg/mL or for solutions with chiral buffer components that absorb far UV light. Infrared (IR) protein absorbance spectra of the Amide I region (1,600–1700 cm−1) contain information about secondary structure and require higher concentrations than circular dichroism often with complementary spectral windows. In this paper, we consider a number of approaches to extract structural information from a protein infrared spectrum and determine their reliability for regulatory and research purpose. In particular, we compare direct and second derivative band-fitting with a self-organising map (SOM) approach applied to a number of different reference sets. The self-organising map (SOM) approach proved significantly more accurate than the band-fitting approaches for solution spectra. As there is no validated benchmark method available for infrared structure fitting, SOMSpec was implemented in a leave-one-out validation (LOOV) approach for solid-state transmission and thin-film attenuated total reflectance (ATR) reference sets. We then tested SOMSpec and the thin-film ATR reference set against 68 solution spectra and found the average prediction error for helix (α + 310) and β-sheet was less than 6% for proteins with less than 40% helix. This is quantitatively better than other available approaches. The visual output format of SOMSpec aids identification of poor predictions. We also demonstrated how to convert aqueous ATR spectra to and from transmission spectra for structure fitting. Fourier self-deconvolution did not improve the average structure predictions

Analysis of secondary structure of proteins by vibrational spectroscopy and self-organizing maps

Antibodies are proteins produced by the immune system and one of the top biopharmaceutical market types due to their applications in oncology therapy among others4. As any other protein, their functionality depends on the preservation of their native form which, under certain stressing conditions, can undergo changes at different structural levels and thus loss of their activity. Although mass spectrometry is a powerful technique for primary structure determination, it often fails to give information at higher order levels. In this project we explored the possibilities of vibrational spectroscopic techniques as a tool kit to help ensure the integrity and batch to batch reproducibility in antibody manufacture. Infrared (IR) and Raman spectra are well known to contain bands (Amide I, II and III) with shapes that correlate to secondary structure (SS)6,7. Unlike Circular Dichroism (CD) (the most well-established technique for secondary structure analysis8), IR and Raman spectroscopy allow much wider ranges of optical density which makes the analysis of complex pharmaceutical samples more feasible. However, the data processing and extraction of this information are ambiguous and, in many cases, limited by spectral noise and water absorption in IR and fluorescence in Raman. In this work, data sets of proteins with known SS were collected in both solid and aqueous state by Raman, IR and Raman Optical Activity and used along a neural network algorithm called Self-Organizing Maps (SOMspec) for SS prediction of proteins. It was found that Raman spectroscopy provides the best predictions followed by IR based on the shape of the amide I band. Although the ROA amide I of proteins has also been reported in the literature to correlate to SS content, we failed to predict SS structure by ROA-SOM based on this band. More work needs to be carried out in the future to attempt to predict SS based on the ROA amides II and III instead

Exploring the potential of molecular spectroscopy for the detection of post-translational modifications of a stressed biopharmaceutical protein

Background: Proteins are biomolecules that consist of sequences of amino acids (primary structure) which can further interact and cause the backbone to fold into more complex structures (secondary and tertiary structures). Any chemical alterations of the molecules after the translation of the messenger RNA code into a protein primary sequence are known as post-translational modifications (PTMs). PTMs may affect the protein’s functionality; thus it is necessary to identify them. PTMs of particular interest to the pharmaceutical industry include deamidation, oxidation, deglycosylation and isomerization, which may occur due to environmental stressors. However, they have proved challenging to identify quickly. Electronic and vibrational spectroscopies have proved valuable tools for studying higher-order structure and stability of proteins. Materials & Methods: In this work, circular dichroism (CD), infrared absorbance (IR) and Raman spectroscopies were applied to characterize antibody (mAb NIP 228) PTMs as a result of different stressors. Mass spectrometry was used to confirm the identity of modifications including the targeted ones. Room temperature CD showed that the secondary structure was the same after all treatments, and temperature-controlled CD showed how protein stability was affected by modifications. Both Raman and IR analysis detected small differences between the reference and deglycosylated proteins, and clearly indicated the presence of other PTMs. Conclusion: This work required some novel computational approaches to pre–process Raman and IR spectra and a review of the band assignments for proteins existing in the literature

Reduction of Background Fluorescence from Impurities in Protein Samples for Raman Spectroscopy

Background fluorescence remains the biggest challenge in Raman spectroscopy because of the consequent curvature of the baseline and the degradation of the signal-to-noise ratio of the Raman signal. While the concentrations of the fluorophore impurities are usually too low to be detected by other analytical methods, they are often sufficient to prevent Raman data collection. Among the different existing methods to remove the fluorescence signal, photobleaching remains the most popular due to its simplicity. However, using the spectrometer laser to photobleach is far from optimal. Most commercially available instruments have little or no choice of wavelength, and their output powers are in many cases not suitable for highly fluorescent samples such as those from biological systems (e.g., proteins). In this article, we assess practical aspects of photobleaching such as the apparent reversibility of the process and the effect of convection currents due to what we speculate to be temperature gradients across the bulk of the solution. We also introduce an affordable custom made external photobleaching unit with a choice of excitation wavelength and demonstrate its viability with a highly fluorescent bovine serum albumin protein solution, which had proved most challenging for Raman spectroscopy as it contained ∼10% w/w impurities

Reduction of background fluorescence of protein samples for Raman spectroscopy

Background fluorescence remains the biggest challenge in Raman spectroscopy because of the consequent curvature of the baseline and the degradation of the signal to noise ratio of the Raman signal. While the concentrations of the fluorophore impurities are usually too low to be detected by other analytical methods, they are often sufficient to prevent Raman data collection. Among the different existing methods to remove the fluorescence signal, photobleaching remains the most popular due to its simplicity. However, using the spectrometer laser to photobleach is far from optimal. Most commercially available instruments have little or no choice of wavelength, and their output powers are in many cases not suitable for highly fluorescent samples such as those from biological systems (e.g., proteins). In this article, we assess practical aspects of photobleaching such as the apparent reversibility of the process and the effect of convection currents due to what we speculate to be temperature gradients across the bulk of the solution. We also introduce an affordable custom made external photobleaching unit with a choice of excitation wavelength and demonstrate its viability with a highly fluorescent bovine serum albumin protein solution, which had proved most challenging for Raman spectroscopy as it contained ~10% w/w impurities