%0 Journal Article
%T Thermal Aggregation of Recombinant Protective Antigen: Aggregate Morphology and Growth Rate
%A Daniel J. Belton
%A Aline F. Miller
%J Journal of Biophysics
%D 2013
%I Hindawi Publishing Corporation
%R 10.1155/2013/751091
%X The thermal aggregation of the biopharmaceutical protein recombinant protective antigen (rPA) has been explored, and the associated kinetics and thermodynamic parameters have been extracted using optical and environmental scanning electron microscopies (ESEMs) and ultraviolet light scattering spectroscopy (UV-LSS). Visual observations and turbidity measurements provided an overall picture of the aggregation process, suggesting a two-step mechanism. Microscopy was used to examine the structure of aggregates, revealing an open morphology formed by the clustering of the microscopic aggregate particles. UV-LSS was used and developed to elucidate the growth rate of these particles, which formed in the first stage of the aggregation process. Their growth rate is observed to be high initially, before falling to converge on a final size that correlates with the ESEM data. The results suggest that the particle growth rate is limited by rPA monomer concentration, and by obtaining data over a range of incubation temperatures, an approach was developed to model the aggregation kinetics and extract the rate constants and the temperature dependence of aggregation. In doing so, we quantified the susceptibility of rPA aggregation under different temperature and environmental conditions and moreover demonstrated a novel use of UV spectrometry to monitor the particle aggregation quantitatively, in situ, in a nondestructive and time-resolved manner. 1. Introduction The study of protein aggregation is a burgeoning field of research driven by the urgent need to elucidate the mechanism of neurodegenerative diseases, the desire to understand and mimic naturesĄŻ ability to create hierarchical complex nanostructures, and the necessity to understand and minimise product loss during the processing and formulation of biopharmaceuticals. Aggregation is of particular importance for therapeutic proteins as it can lead to a loss of product, reduce efficacy, alter biological activity and pharmacokinetics, and even raise safety concerns such as increased immunogenicity [1¨C3]. Aggregation can be induced by solution conditions such as protein concentration, pH, salinity, temperature, and the presence of additives [4, 5]. These variables are also known to affect the quantity and morphology of aggregate formed [5]. Stresses to the protein such as over-expression, refolding, freeze-thaw cycles, agitation, or exposure to hydrophobic surfaces or air (including foaming) can also lead to the formation of aggregates [2, 4¨C6]. Each of these environmental factors is typically encountered during
%U http://www.hindawi.com/journals/jbp/2013/751091/