Solvent‐free liquid avidin as a step toward cold chain elimination

Abstract

The temperature sensitivity of vaccines and therapeutic proteins forces the distribution of life‐saving treatments to rely heavily on the temperature‐controlled (usually 2–8°C) supply and distribution network known as the cold chain. Here, using avidin as a model, we demonstrate how surface engineering could significantly increase the thermal stability of therapeutic proteins. A combination of spectroscopic (Fourier transform infrared, circular dichroism, and ultraviolet‐visible) and scattering techniques (dynamic light scattering, small‐angle, and wide‐angle X‐ray scattering) were deployed to probe the activity, structure, and stability of the model protein. Temperature‐dependent synchrotron radiation circular dichroism spectroscopy was used to demonstrate a significant increase in thermal stability, with a half denaturation temperature of 139.0°C and reversible unfolding with modified avidin returning to a 90% folded state when heated to temperatures below 100°C. Accelerated aging studies revealed that modified avidin retained its secondary structure after storage at 40°C for 56 days, equivalent to 160 days at 25°C. Furthermore, binding studies with multiple ligands revealed that the binding site remained functional after modification. As a result, this approach has potential as a storage technology for therapeutic proteins and the elimination of the cold chain, enabling the dissemination of life‐saving vaccines worldwide.

Recovery of enzyme structure and activity following rehydration from ionic liquid

“…This is an important finding given the current interest in IL-based storage media to ''break the cold chain'' for protein therapeutics. 36 Activity data and simulations both show that lysozyme's enzymatic function is suppressed at high IL content but is recovered upon sufficient rehydration with water. From simulations it is observed that [EMIM + ] ions occupying the ligand position and the vicinity of the active site.…”

“…This is an important finding given the current interest in IL-based storage media to “break the cold chain” for protein therapeutics. 36…”

Synthesis and functionalization of dendritic polyglycerol-based nanogels: application in T cell activation

“…CD spectroscopy revealed that the secondary structure of Av was still intact after coupling (Figure 3C). 47 Cryo-TEM images of NG-Av 12 (Figure 3D) show spherical particles with a rather narrow size distribution. Just as for the measurements for the Nanogel 9 (Figure 1D), a smaller diameter as compared to DLS can be observed, which again can be attributed to the missing hydration shell.…”

Molecular simulations explain the exceptional thermal stability, solvent tolerance and solubility of protein–polymer surfactant bioconjugates in ionic liquids

“…Solvent-free protein liquids (SFPLs) are a class of hybrid viscous materials, prepared by cationization of surface-exposed aspartate and glutamate residues followed by coupling the positively charged surface sites with anionic surfactants, and solvent removal. [5][6][7] Many proteins have been thus conjugated with polymeric surfactants and studied using several experimental approaches [8][9][10][11][12][13][14][15][16][17][18] and a few computational ones. [19][20][21][22][23] Proteins are significantly more thermostable as SFPLs than as conventional aqueous solutions, i.e., they retain their secondary structure and can be functional even beyond 100 1C.…”