Analytical Techniques for Structural Characterization of Proteins in Solid Pharmaceutical Forms: An Overview

Abstract

Therapeutic proteins as biopharmaceuticals have emerged as a very important class of drugs for the treatment of many diseases. However, they are less stable compared to conventional pharmaceuticals. Their long-term stability in solid forms, which is critical for product performance, depends heavily on the retention of the native protein structure during the lyophilization (freeze-drying) process and, thereafter, in the solid state. Indeed, the biological function of proteins is directly related to the tertiary and secondary structure. Besides physical stability and biological activity, conformational stability (three-dimensional structure) is another important aspect when dealing with protein pharmaceuticals. Moreover, denaturation as loss of higher order structure is often a precursor to aggregation or chemical instability. Careful study of the physical and chemical properties of proteins in the dried state is therefore critical during biopharmaceutical drug development to deliver a final drug product with built-in quality that is safe, high-quality, efficient, and affordable for patients. This review provides an overview of common analytical techniques suitable for characterizing pharmaceutical protein powders, providing structural, and conformational information, as well as insights into dynamics. Such information can be very useful in formulation development, where selecting the best formulation for the drug can be quite a challenge.

Impact of process stress on protein stability in highly-loaded solid protein/PEG formulations from small-scale melt extrusion

“…The CD spectra of all BSA samples were mostly overlapping, however in the FTIR spectra some shifts are shown. A probable explanation for the observed differences in the FTIR spectra at 1652 cm −1 may be the extrusion procedure, since extrusion (thermal and shear stress) can cause reversible changes in protein conformation and is associated with a reduction in the α-helical content and an increase in the random chain content ( Bolje and Gobec, 2021 ; Bagińska et al, 2008 ). Human insulin was also studied for changes in its secondary structure as a result of temperature and shear stress during extrusion processing.…”

“…These changes indicated the transformation of α-helix content to β-sheets or a disordered structure which enhances the tendency to aggregate. In particular, the band at 1652 cm −1 which corresponds to α-helical structures ( Bolje and Gobec, 2021 ), was obviously affected by TSE with screws containing a kneading element due to higher shear stress and residence time. As can be seen in the CD and FTIR spectra, ram extrusion had the lowest impact on secondary structure changes of human insulin ( Fig.…”

Protein Function Analysis through Machine Learning

“…The protein structure initiative (PSI) of the early 2000s aimed to distribute 3D structural information for naturally occurring proteins through experimental methods such as: (i) X-ray crystallography (XRC); (ii) nuclear magnetic resonance (NMR); (iii) cryo-electron microscopy (cryo-EM), each with its advantages and disadvantages [ 64 , 65 ]. Other available experimental methods include SAXS, CD, FRET, and Raman spectroscopy [ 66 ]. Notably, many protein structures are resolved when bound to a ligand.…”

Development of a protective lyophilisation medium and conditions to stabilise the erythrocyte diagnostic preparation of tularaemia immunoglobulin

“…Поэтому в качестве эффективных лиопротекторов использовали дисахариды, такие как трегалоза и сахароза. Некоторыми исследователями было обнаружено, что добавление сахарозы к препаратам при лиофилизации приводит к сдвигу температуры плавления в сторону увеличения, обеспечивая таким образом эффективную защиту препарата [20,21]. Для лиофилизации в составе защитных сред также успешно применяются коллоиды (желатин, агар, пептон, молоко и сыворотки).…”