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Effect of Azide Preservative on Thermomechanical Aggregation of Purified Reference Protein Materials

Published

Author(s)

Sean Lehman, Ioannis Karageorgos, Wyatt N. Vreeland, Jeremy Filteau

Abstract

Protein aggregation can affect the quality of protein-based therapeutics and is an important aspect of disease progression in multiple illnesses. Attempting to unravel the mechanisms and factors influencing protein aggregation involves systematic studies. But these studies often include sodium azide or similar preservatives in the aggregation buffer. This work shows the effects of azide on aggregation of two highly purified reference proteins, both a bovine serum albumin (BSA) as well as the NIST monoclonal antibody (NISTmAb). The proteins were aggregated by a combination of thermal and mechanical stress in a controlled manner to prepare soluble sub-micrometer protein aggregates. Aggregates were characterized over time and across concentrations of sodium azide in their respective buffers. Protein particles were characterized by field flow fractionation with light scattering measurements. The light scattering analysis along with quantification by UV spectroscopy reveals strong time-dependent generation of aggregated protein and a commensurate increase in aggregate molar mass, both of which depend on azide concentration. Gel electrophoresis was used to probe the reversibility of the aggregation and demonstrated complete reversibility for the NISTmAb, yet to a lesser extent for the BSA. Kinetic fitting to a commonly implemented nucleated polymerization model was also employed for the NISTmAb 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 clinically relevant proteins.
Citation
Journal of Pharmaceutical Sciences
Volume
110
Issue
5

Keywords

protein aggregation, light scattering, modeling, protein electrophoresis, aggregation mechanism

Citation

Lehman, S. , Karageorgos, I. , Vreeland, W. and Filteau, J. (2021), Effect of Azide Preservative on Thermomechanical Aggregation of Purified Reference Protein Materials, Journal of Pharmaceutical Sciences, [online], https://doi.org/10.1016/j.xphs.2021.01.013 (Accessed April 14, 2024)
Created January 12, 2021, Updated October 7, 2022