Impact of Sucrose Level on Storage Stability of Proteins in Freeze-Dried Solids: II. Correlation of Aggregation Rate with Protein Structure and Molecular Mobility
Bingquan S. Wang, Serguei Tchessalov, Marcus T. Cicerone, Nicholas W. Warne, Michael Pikal
The purpose of this study is to investigate the impact of sucrose level on storage stability of dried proteins and thus better understand the mechanism of protein stabilization by disaccharides in lyophilized products. Three IgG1 fusion proteins and two cytokines were formulated with different amounts of sucrose and then freeze dried below the collapse temperature. Protein aggregation, following storage at (25, 40 and 50) °C, was quantified using Size Exclusion Chromatography. Protein secondary structure upon lyophilization was monitored by Fourier Transform Infrared (FTIR). The rate of enthalpy relaxation, a measure of global molecular mobility, was studied using Thermal Activity Monitor (TAM). Fast local dynamics with a timescale of nanoseconds was characterized by neutron backscattering. The density of the dried protein formulations was measured with a gas pycnometer. Both FTIR structure and structural relaxation time constant (t²) from TAM achieved maxima at about 1/1 mass ratio for most proteins studied. The physical stability of the proteins, on the other hand, increased monotonically with an increasing content of sucrose in the formulation over the entire range of compositions studied, indicating a contribution to stability beyond maintenance of native conformation. Both the fast local mobility and free volume obtained from density decreased monotonically with an increased level of sucrose in the protein formulations. Protein stabilization by sugar, therefore, cannot be completely explained by global dynamics (i.e. tau² from TAM) and FTIR structure throughout the whole range of compositions studied. Instead, the fast local dynamics and free volume correlate well with protein storage stability. Local glass dynamics seems to be more important than protein secondary structure to the storage stability of protein in the protein/sucrose systems at temperatures well below Tg.
Protein stability, aggregation, stabilizer, freeze-drying, lyophilization, glass dynamics, molecular mobility, fast dynamics, local motion, protein structure, specific interaction, protein-sugar interaction, water substitute, structural relaxation, free volume, protein stabilization, fusion protein, cytokine
, Tchessalov, S.
, Cicerone, M.
, Warne, N.
and Pikal, M.
Impact of Sucrose Level on Storage Stability of Proteins in Freeze-Dried Solids: II. Correlation of Aggregation Rate with Protein Structure and Molecular Mobility, Journal of Pharmaceutical Sciences, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=854104
(Accessed December 2, 2023)