Nano Differential Scanning Fluorimetry-Based Thermal Stability Screening and Optimal Buffer Selection for Immunoglobulin G

Abstract

Nano differential scanning fluorimetry (nanoDSF) is a high-throughput protein stability screening technique that simultaneously monitors protein unfolding and aggregation properties. The thermal stability of immunoglobulin G (IgG) was investigated in three different buffers (sodium acetate, sodium citrate, and sodium phosphate) ranging from pH 4 to 8. In all three buffers, the midpoint temperature of thermal unfolding (Tm) showed a tendency to increase as the pH increased, but the aggregation propensity was different depending on the buffer species. The best stability against aggregation was obtained in the sodium acetate buffers below pH 4.6. On the other hand, IgG in the sodium citrate buffer had higher aggregation and viscosity than in the sodium acetate buffer at the same pH. Difference of aggregation between acetate and citrate buffers at the same pH could be explained by a protein–protein interaction study, performed with dynamic light scattering, which suggested that intermolecular interaction is attractive in citrate buffer but repulsive in acetate buffer. In conclusion, this study indicates that the sodium acetate buffer at pH 4.6 is suitable for IgG formulation, and the nanoDSF method is a powerful tool for thermal stability screening and optimal buffer selection in antibody formulations.

Biochemical and Structural Characterization of OvoATh2: A Mononuclear Nonheme Iron Enzyme from Hydrogenimonas thermophila for Ovothiol Biosynthesis

“…Analysis of the protein domain organization of OvoA Th2 suggests that similar to that of OvoA Eta , OvoA Th2 has both sulfoxide synthase and methyltransferase domains (Figure S1). Due to thermostability concerns in OvoA crystallization, after overexpression and purification of OvoA Th2 (Figure S2) using a protocol similar to those employed for OvoA Eta and OvoA Mtht characterization, , we assessed the thermostability of OvoA Th2 using a nanoDSF assay (Figure B). , The thermal unfolding curves of the peptide of OvoA Mtht are included for comparison purposes. Our analysis demonstrated that the T M of OvoA Th2 is approximately 21 °C higher than that of OvoA Mtht (Figure B).…”

Enhancing viscosity control in antibody formulations: A framework for the biophysical screening of mutations targeting solvent-accessible hydrophobic and electrostatic patches

“…Thermal differential scanning fluorimetry (DSF) measurements were performed using a Prometheus NT.48 (NanoTemper Technologies, Germany) equipped with back-reflection technology for high-throughput analysis of unfolding temperature (T m ), calculated from the intrinsic fluorescence intensity ratio of tyrosine and tryptophan (350/330 nm). 40 Prior to each experiment, the excitation power was set to achieve ≥5,000 counts in the discovery scan. Corresponding profiles were analysed in Prometheus NT.48 and the first derivative calculated.…”

A first insight into the developability of an IgG3: A combined computational and experimental approach

“…The intrinsic fluorescence from unfolding events exposing tyrosine and tryptophan residues were monitored via the 350/330 nm intensity ratio. 12 A temperature ramp of 2°C/minute from 20-95 °C was performed. Both samples were assessed at concentrations ~150 mg/mL and unfolding temperatures of antibody domains (T m1 , T m2 and T m3 ) detected from first-derivative peaks of the 350/330 nm fluorescence intensity ratio.…”