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Quantifying protein aggregation kinetics using electrospray differential mobility analysis



Kaleb J. Duelge, Jeremie Parot, Vincent A. Hackley, Michael R. Zachariah


Protein aggregation is a significant concern in bioprocessing and end use applications. Here the thermal aggregation kinetics of bovine serum albumin (BSA) and α-chymotrypsinogen A (α- chymo) were measured over a range of concentrations and temperatures by two orthogonal monomer- loss techniques: electrospray differential mobility analysis (ES-DMA) and asymmetrical flow field flow fractionation (AF4). Both instruments were able to successfully resolve the protein monomer and dimer. The aggregation was found to be second order for BSA and first order for α- chymo, suggesting significantly different limiting steps of aggregation for the two proteins. This is the first reported analysis of the effects of different conditions on protein aggregation kinetics by ES-DMA and the first direct comparison between ES-DMA and AF4 for aggregation kinetics. Temperature-dependent rates were analyzed using the Arrhenius equation to estimate the activation energy of protein aggregation. The ES-DMA determined rates were slightly faster than those by AF4, so instrumental differences were analyzed to identify potential causes. AF4 measured the kinetics at the mg mL-1 level while ES-DMA was sensitive to μg mL-1 concentrations. The limitations of each method demonstrate the importance of orthogonal measurement approaches for the analysis of protein kinetics. These techniques can provide useful alternatives to size exclusion chromatography to screen the stability of formulation conditions for protein therapeutics, as ES-DMA and AF4 do not rely on column interactions for separation and can measure samples in lower ionic strength buffers.
Journal of Pharmaceutical and Biomedical Analysis


Aggregation, kinetics, differential mobility analysis, field flow fractionation, bovine serum albumin


Duelge, K. , Parot, J. , Hackley, V. and Zachariah, M. (2020), Quantifying protein aggregation kinetics using electrospray differential mobility analysis, Journal of Pharmaceutical and Biomedical Analysis, [online], (Accessed May 18, 2024)


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Created January 5, 2020, Updated February 10, 2020