Kinetic Pathway Analysis of Aggregation of Therapeutic Proteins

Thursday, November 11, 2010: 5:25 PM
Grand Ballroom E (Salt Palace Convention Center)
Johanna Wiesbauer, Ulrich Roessl, Stefan Leitgeb and Bernd Nidetzky, TU Graz, Institute of Biotechnology and Biochemical Engineering, Research Center Pharmaceutical Engineering GmbH, Graz, Austria

Protein aggregation represents probably the most common and troubling manifestation of protein instability. Shelf life of therapeutic proteins is particularly impaired by aggregation, which can occur almost in all phases of protein drug development. Administration of protein aggregates may lead to reduced pharmacological activity and adverse drug reactions. Thus, prediction of protein aggregation is of major interest for pharmaceutical industry. Existing sequence based bioinformatic tools are barely able to predict the aggregation behavior of therapeutic proteins. To implement new approaches for this, detailed analysis of protein aggregation, its underlying mechanisms and its kinetics is necessary. A very important group of therapeutic proteins are the cytokines which play a prominent role in cell signaling and communication. In our study we simulate process conditions for induction of aggregation of two cytokines of the four α-helix bundle family as reference model systems. The experiments were carried out under accelerated aggregation conditions since protein aggregation is usually a slow process and often leads to aggregates in the percent or permille range. Even though this is not enough for detailed characterization it may be enough to trigger immune reactions. The resulting aggregates were quantified by size exclusion HPLC. Identification of different aggregation intermediates under certain process and formulation conditions over time enabled kinetic pathway analysis. Further characterization was accomplished using intrinsic fluorescence, polyacrylamid gel electrophoresis and mass spectrometry.

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