The effect of deuterium oxide on the conformational stability and aggregation of bovine serum albumin

Abstract

Protein aggregation is a significant problem affecting the integrity of proteins, and is a major hindrance to the development of biopharmaceutical products. Deuterium oxide (DO), widely used in protein characterization studies, has been shown to promote protein aggregation when used as a substitute for water in most buffered protein solutions; however, a few studies have reported minor improvements in melting point temperatures for some proteins. Our study aims to investigate the effect of DO on protein stability, using bovine serum albumin (BSA) as a model. We performed accelerated stability studies at high temperatures and assessed the physical and conformational stability of BSA using fluorescence spectroscopy, dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography. Our findings reveal that DO enhances the conformational stability of monomeric BSA, reducing monomer loss and formation of small aggregates at high temperatures. There is also an increase in the formation of larger aggregates probed by thioflavin T (ThT), however, the increase is not considered significant based on DLS results. Our findings demonstrate that exchanging water with DO can improve the stability of proteins in solution, by maintaining the stability of the monomeric form, which may be beneficial for the long-term storage of some biological products.

Enthalpic stabilization of an SH3 domain by D2O

“…D 2 O affects protein–carbohydrate, protein–peptide, and protein–nucleic acid interactions, with effects on the enthalpy of binding. In addition to binding, D 2 O enhances protein oligomerization and aggregation, by what has been suggested to be the promotion of hydrophobic interactions. Given the influence of heavy water on biomolecular reactions, D 2 O is also expected to affect whole organisms.…”

“…D 2 O affects protein-carbohydrate, protein-peptide, and protein-nucleic acid interactions, 42 with effects on the enthalpy of binding. In addition to binding, D 2 O enhances protein oligomerization and aggregation, 36,[43][44][45][46][47][48][49][50][51][52] by what has been suggested to be the promotion of hydrophobic interactions. Given the influence of heavy water on biomolecular reactions, 33 Enzyme assay Stabilizing Residual enzyme activity Staphylococcal nuclease 34 Urea, CD, FTIR Stabilizing m-Value, ΔG ' Domain 1 of rat CD2 35 GdnHCl, stopped-flow fluorescence spectroscopy 22 DSC Stabilizing T m , ΔG 0 , ΔH 0 , ΔS 0 , ΔC p ' Bovine serum albumin 22 DSC Stabilizing T m , ΔG 0 , ΔH 0 , ΔS 0 , ΔC p ' Bovine serum albumin 39 Heat, far-UV CD Stabilizing Molar ellipticity D 2 O is also expected to affect whole organisms.…”

“…64,65 More recent studies show that low concentrations may be necessary and even beneficial, 66,67 with interesting recent hypotheses on the use of heavy isotopes for increasing human longevity. 68 Finally, there is some interest in the use of D 2 O as an excipient 52,69 because of the observation that D 2 O can stabilize vaccines. 70 Understanding the effects of isotopic waters is key to understanding biology, including protein folding.…”

The decay of the refocused Hahn echo in double electron–electron resonance (DEER) experiments

“…where V 3 is the term cubic in b mn , and we have collected all terms of orders higher than 2 in b nm into a single term, f . This factorization is referred to as the linked-cluster expansion (Saikin et al, 2007).…”

Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

“…For example, polysorbates are commonly used in biologics as a stabilizing agent; however, their addition in high concentrations can denature proteins and cause adverse side effects such as injection site reactions [114,115,116]. Injectable mAb formulations are co-formulated with recombinant human hyaluronidase, specifically as a permeation enhancer for more efficient absorption into tissue, although the inclusion of this additional biologic adds further burden to the formulation’s viscosity and propensity to aggregate [5,8,25,26,27,28,117,118,119,120,121,122,123]. Antibody therapies for IV administration are prepared as lyophilised powder for reconstitution and further dilution, and injectable administrations are prepared as liquid-based formulations in pre-filled syringes.…”