Fourier transform infared spectroscopy investigation of protein conformation in spray‐dried protein/trehalose powders

Abstract

Spray drying is a way to generate protein solids (powders), which is also true for lyophilization. Sugars are used to protect proteins from conformational changes and chemical degradations arising from drying processes and storage conditions such as the humidity. The influence of trehalose and humidity on the conformation and hydration of spray-dried recombinant human granolucyte colony stimulating factor (rhG-CSF) and recombinant consensus interferon-alpha (rConIFN) was investigated using Fourier transform IR spectroscopy. The spectral analysis of spray-dried powders in the amide I region demonstrated that trehalose stabilized the alpha-helical conformation of both rhG-CSF and rConIFN proteins. Exposure of the pure protein powders to 33% relative humidity (RH) resulted in the formation of beta sheets and loss of turns but no change in alpha-helical structure. Trehalose reduced the magnitude of the changes in beta sheets and turns. Exposure of the pure protein powders to 75% RH resulted in the loss of alpha-helical conformation with a corresponding increase in beta structures (beta sheets and turns). Trehalose did not protect proteins from the loss of alpha-helical structures, but it reduced the formation of antiparallel beta sheets. Hydrogen-deuterium exchange (H-D exchange) was used to further characterize these hydration-induced conformational changes. At 33% RH the percent exchange of the protein decreased with increasing trehalose content, indicating a greater protection of the protein from H-D exchange by a higher concentration of trehalose. Such protection correlates with decreased conformational changes of the protein by trehalose at this humidity. At 75% RH the degree of H-D exchange of the protein was insensitive to the powder composition in all powders. Surprisingly, the H-D exchange of trehalose was low at about 20-25%, which was nearly independent of the protein/trehalose ratio and humidity, indicating that the exchangeable protons on trehalose molecules are highly protected in protein-containing powders. The observed protein hydration is related to the effect of trehalose on the conformational changes of the protein under humidity.

Trehalose and calcium exert site‐specific effects on calmodulin conformation in amorphous solids

“…Such information is not available by rapid spectroscopic methods used to characterize amorphous solids (e.g., FTIR, Raman), which produce signals that correspond to functional groups rather than to specific protein structural regions, and is timeconsuming to acquire by solid-state NMR, a method which also requires isotopically labeled protein. Previous studies of H/D exchange in lyophilized proteins have employed analysis by FTIR (French et al, 2004), a technique that provides information on particular functional groups but cannot provide resolution at the amino acid or peptide level.…”

Controlled enzymatic hydrolysis on characteristic and antioxidant properties of soybean protein isolate-maltodextrin conjugates

“…The protein samples were prepared using potassium bromide (KBr) pellet method. [20] Infrared spectra were measured with a Fourier transform infrared spectrophotometer (Nicolet iS10) (Thermo, USA) at 20°C. Full-wave scanning was collected with 32 scans in the 4000-400 cm −1 region.…”

Protein Conformation in Amorphous Solids by FTIR and by Hydrogen/Deuterium Exchange with Mass Spectrometry

“…Generally, lyophilized formulations are exposed to D 2 O vapor for variable lengths of time at a predetermined relative humidity (RH) value before being analyzed by a suitable technique. French et al (7) used HDX with FTIR analysis to characterize human granulocyte colony stimulating factor (rhG-CSF) and recombinant consensus interferon-a (rConIFN) in spray-dried powders containing trehalose, using isotopic shifts in the amide II/II9 bands. Desai et al (8) employed HDX with 1 H NMR analysis to study the unfolding of bovine pancreatic trypsin inhibitor (BPTI) on lyophilization.…”