Protein aggregation can affect the quality of protein-based therapeutics. Attempting to unravel factors influencing protein aggregation involves systematic studies. These studies often include sodium azide or similar preservatives in the aggregation buffer. This work shows effects of azide on aggregation of two highly purified reference proteins, both a bovine serum albumin (BSA) as well as a monoclonal antibody (NISTmAb). The proteins were aggregated by thermomechanical stress, consisting of simultaneous heating of the solution with gentle agitation. Protein aggregates were characterized by asymmetric flow field flow fractionation (AF4) with light scattering measurements along with quantification by UV spectroscopy, revealing strong time-dependent generation of aggregated protein and an increase in aggregate molar mass. Gel electrophoresis was used to probe the reversibility of the aggregation and demonstrated complete reversibility for the NISTmAb, but not so for the BSA. Kinetic fitting to a commonly implemented nucleated polymerization model was also employed to provide mechanistic details into the kinetic process. The model suggests that the aggregation of the NISTmAb proceeds via nucleated growth and aggregate-aggregate condensation in a way that is dependent on the concentration (and presence) of the azide anion. This work overall implicates azide preservatives as having demonstrable effects on thermomechanical stress and aggregation of proteins undergoing systematic aggregation and stability studies.
Keywords: Field-flow fractionation (FFF); Light scattering (static); Mechanistic modeling; Monoclonal antibody; Protein aggregation.
Published by Elsevier Inc.