Quantifying protein aggregation kinetics using electrospray differential mobility analysis

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Abstract

Protein aggregation is a critical concern in bioprocessing, where its presence can result in serious adverse interactions in clinical end-use applications. In this study, an aerosol-based technique, electrospray differential mobility analysis (ES-DMA), was used to quantify thermally-induced protein aggregation kinetics for bovine serum albumin (BSA) and α-chymotrypsinogen A (α-chymo), employing a new methodology to modify the solution for compatibility with the electrospray process. Results are compared orthogonally with asymmetrical-flow field-flow fractionation (AF4), a hydrodynamic separation technique with UV detection. Measurements were conducted over a range of protein concentrations and temperatures. Both techniques successfully resolved the protein monomer and dimer populations, allowing quantification of monomer loss. BSA and α-chymo exhibited second and first order kinetics, respectively, confirming different limiting steps for the two species. The Arrhenius equation yielded activation energies for BSA of (240 ± 20) kJ mol−1 and (190 ± 10) kJ mol−1 by ES-DMA and AF4, respectively. The rates determined by ES-DMA were equal to or slightly faster than those measured by AF4, so instrumental differences were analyzed to identify potential sources of bias. An important factor may be the applicable concentration range for each method; notably, AF4 operates at the mg mL-1 level, while ES-DMA is sensitive at μg mL-1 and therefore requires much smaller samples for analysis (typically several μL are injected). The limitations of each method are detailed in the discussion and demonstrate the importance of orthogonal measurement strategies for the analysis of protein kinetics. ES-DMA provides a potentially useful alternative to size exclusion chromatography to screen the stability of formulation conditions for protein therapeutics; neither ES-DMA nor AF4 rely on column interactions for separation.

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Duelge, K. J., Parot, J., Hackley, V. A., & Zachariah, M. R. (2020). Quantifying protein aggregation kinetics using electrospray differential mobility analysis. Journal of Pharmaceutical and Biomedical Analysis, 177. https://doi.org/10.1016/j.jpba.2019.112845

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