Proteins and antibodies in serum, plasma, and whole blood—size characterization using asymmetrical flow field-flow fractionation (AF4)

The analysis of aggregates of therapeutic proteins is crucial in order to ensure efficacy and patient safety. Typically, the analysis is performed in the finished formulation to ensure that aggregates are not present. An important question is, however, what happens to therapeutic proteins, with regard to oligomerization and aggregation, after they have been administrated (i.e., in the blood). In this paper, the separation of whole blood, plasma, and serum is shown using asymmetric flow field-flow fractionation (AF4) with a minimum of sample pre-treatment. Furthermore, the analysis and size characterization of a fluorescent antibody in blood plasma using AF4 are demonstrated. The results show the suitability and strength of AF4 for blood analysis and open new important routes for the analysis and characterization of therapeutic proteins in the blood. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s00216-018-1127-2) contains supplementary material, which is available to authorized users.