Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) Spectroscopy as an Analytical Method to Investigate the Secondary Structure of a Model Protein Embedded in Solid Lipid Matrices

Abstract

Protein drugs may encounter conformational perturbations during the formulation processing of lipid-based solid dosage forms. In aqueous protein solutions, attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy can investigate these conformational changes following the subtraction of spectral interference of solvent with protein amide I bands. However, in solid dosage forms, the possible spectral contribution of lipid carriers to protein amide I band may be an obstacle to determine conformational alterations. The objective of this study was to develop an ATR FT-IR spectroscopic method for the analysis of protein secondary structure embedded in solid lipid matrices. Bovine serum albumin (BSA) was chosen as a model protein, while Precirol AT05 (glycerol palmitostearate, melting point 58 ℃) was employed as the model lipid matrix. Bovine serum albumin was incorporated into lipid using physical mixing, melting and mixing, or wet granulation mixing methods. Attenuated total reflection FT-IR spectroscopy and size exclusion chromatography (SEC) were performed for the analysis of BSA secondary structure and its dissolution in aqueous media, respectively. The results showed significant interference of Precirol ATO5 with BSA amide I band which was subtracted up to 90% w/w lipid content to analyze BSA secondary structure. In addition, ATR FT-IR spectroscopy also detected thermally denatured BSA solid alone and in the presence of lipid matrix indicating its suitability for the detection of denatured protein solids in lipid matrices. Despite being in the solid state, conformational changes occurred to BSA upon incorporation into solid lipid matrices. However, the extent of these conformational alterations was found to be dependent on the mixing method employed as indicated by area overlap calculations. For instance, the melting and mixing method imparted negligible effect on BSA secondary structure, whereas the wet granulation mixing method promoted more changes. Size exclusion chromatography analysis depicted the complete dissolution of BSA in the aqueous media employed in the wet granulation method. In conclusion, an ATR FT-IR spectroscopic method was successfully developed to investigate BSA secondary structure in solid lipid matrices following the subtraction of lipid spectral interference. The ATR FT-IR spectroscopy could further be applied to investigate the secondary structure perturbations of therapeutic proteins during their formulation development.

ATR-FTIR spectroscopy and spectroscopic imaging for the analysis of biopharmaceuticals

“…This research managed to observe the binding of a single phosphate on ATPase using a combination of ATR-FTIR spectroscopy with biospecific interaction analysis (BIA)-ATR biosensors, which allows the surface of the IRE to adsorb to a biomembrane [74]. ATR-FTIR spectroscopy has been used to investigate the secondary structure of denatured bovine serum albumin (BSA) alone and in a mixture with a native protein solid, which could be applied to investigate the solid-state formulation development process more thoroughly [75]. ATR-FTIR spectroscopy has also been used extensively to study proteins, in particular biopharmaceuticals, and exciting research has shown the potential applications of it to investigate and monitor changes effectively in PTMs and secondary structures.…”

Systematic FTIR Spectroscopy Study of the Secondary Structure Changes in Human Serum Albumin under Various Denaturation Conditions

“…The main advantage of applying the IR spectroscopy for the analysis of protein structural characteristics is the independence of this method on the protein molecule size or the physical state of the sample. Not only the hydrated films or solid-state can be easily studied, but also the aqueous solution [12], organic solvents, detergents, micelles, and phospholipid membranes, all these states allow to study the native structure of the protein [13], as well as to assess the effect of the medium on the protein structure.…”

Unveiling the inhibitory mechanism of aureusidin targeting xanthine oxidase by multi-spectroscopic methods and molecular simulations

“…Since FT-IR is less sensitive to liquids than to solids, the protein secondary structure information was obtained by split-peak tting of the spectra results by origin soware. [31][32][33] As shown in Fig. 7, the peak positions shied and the absorbance changed aer the addition of AUR, indicating that the binding of AUR to XO affected the structure of XO protein.…”