Small angle X-ray scattering and molecular dynamic simulations provide molecular insight for stability of recombinant human transferrin

Development of a fast screening method for selecting excipients in formulations using MD simulations, NMR and microscale thermophoresis

“…Previously, we have applied such an in-silico screening method in combination with small angle X-ray scattering to understand protein-excipient interactions and their effect on oligomerization and dynamics. [21,22] Thus, the discussed fast screening methodology can effectively be carried forward to study the effect of combination of different excipients and how it alters proteins dynamics and thus aggregation.…”

“…[18][19][20] We have previously applied such in-silico screening methods in combination with small angle X-ray scattering to map protein-excipient interaction sites and study their effect on dynamics and oligomerization of human transferrin and albumin-neprilysin fusion protein. [21,22] In this study, we introduce an alternative docking approach which is relatively fast in comparison with the already existing approaches and performs blind docking and clustering to identify predominant binding sites. Taking this further in combination with MD simulations and MST, one can predict fairly well the protein-excipient binding sites.…”

Albumin-neprilysin fusion protein: understanding stability using small angle X-ray scattering and molecular dynamic simulations

“…Dialysis and formulation procedure was performed according to the protocol described in Kulakova et al 20 HSA-NEP was provided by AstraZeneca in 49 g/L solution and was dialyzed for pH/ NaCl and buffer/excipients screening. After dialysis, HSA-NEP was diluted to 20 g/L with dialysis buffer, followed by 1:20 dilution with final formulation buffer within pH of ±0.5 20 . The concentration was determined with a NanodropTM 1000 (Thermo Fisher Scientific, Waltham, USA) (see Table S.1 in supplementary information (SI)).…”

“…www.nature.com/scientificreports www.nature.com/scientificreports/ isothermal chemical denaturation. Chemical denaturation studies was on Unchained Labs HUNK system -AVIA ICD 2304 (Unchained Labs, Pleasanton, USA) according to the protocol described in Kulakova et al 20 using 1134 min of additional incubation time. Both urea and guanidine hydrochloride (GuHCl) were used as denaturants for pH/NaCl screening, while urea was selected for the buffer/excipients.…”

“…thermal denaturation. Thermal stability studies were performed in triplicate with the Prometheus NT.48 (NanoTemper Technologies, Munich, Germany) according to the protocol described in Kulakova et al 20 . HSA-NEP stability was analyzed by monitoring the change in T ½ from the first transition.…”