The effects of intramolecular and intermolecular electrostatic repulsions on the stability and aggregation of NISTmAb revealed by HDX‐MS, DSC, and nanoDSF

Citations Over TimeTop 19% of 2021 papers

Abstract

The stability and aggregation of NIST monoclonal antibody (NISTmAb) were investigated by hydrogen/deuterium exchange mass spectrometry (HDX-MS), differential scanning calorimetry (DSC), and nano-differential scanning fluorimetry (nanoDSF). NISTmAb was prepared in eight formulations at four different pHs (pH 5, 6, 7, and 8) in the presence and absence of 150 mM NaCl and analyzed by the three methods. The HDX-MS results showed that NISTmAb is more conformationally stable at a pH near its isoelectric point (pI) in the presence of NaCl than a pH far from its pI in the absence of NaCl. The stabilization effects were global and not localized. The midpoint temperature of protein thermal unfolding transition results also showed the C_H 2 domain of the protein is more conformationally stable at a pH near its pI. On the other hand, the onset of aggregation temperature results showed that NISTmAb is less prone to aggregate at a pH far from its pI, particularly in the absence of NaCl. These seemingly contradicting results, higher conformational stability yet higher aggregation propensity near the pI than far away from the pI, can be explained by intramolecular and intermolecular electrostatic repulsion using Lumry-Eyring model, which separates folding/unfolding equilibrium and aggregation event. The further a pH from the pI, the higher the net charge of the protein. The higher net charge leads to greater intramolecular and intermolecular electrostatic repulsions. The greater intramolecular electrostatic repulsion destabilizes the protein and the greater intermolecular electrostatic repulsion prevents aggregation of the protein molecules at pH far from the pI.

Related Papers

• → Isotopic dependence of intramolecular and intermolecular vibrational couplings in cooperative hydrogen bond networks: singly hydrated phenyl-α-d-mannopyranoside as a case study(2023)6 cited
• → Effect of intermolecular interactions on vibrational-energy transfer in the liquid phase(1993)83 cited
• → A comparison of the intramolecular and intermolecular hydrogen bonding of N,N′-ethylenebis(aminobenzylidene) in the solid state with its salen analogue(2004)15 cited
• → The X-Ray Structure of 5-Chlorosalicylaldehyde(2012)3 cited
• → Dynamic Aspects of Intermolecular Interactions(1998)