Analytical Challenges Assessing Protein Aggregation and Fragmentation Under Physiologic Conditions

Therapeutic proteins are administered by injection or infusion. After administration, the physiologic environment in the desired body compartment - fluid or tissue - can impact protein stability and lead to changes in the safety and/or efficacy profile. For example, protein aggregation and fragmentation are critical quality attributes of the drug product and can occur after administration to patients. In this context, the in vivo stability of therapeutic proteins has gained increasing attention. However, in vivo protein aggregation and fragmentation are difficult to assess and have been rarely investigated. This mini-review summarizes analytical approaches to assess the stability of therapeutic proteins using simulated physiologic conditions. Furthermore, we discuss factors potentially causing in vivo protein aggregation, precipitation, and fragmentation in complex biological fluids. Different analytical approaches are evaluated with respect to their applicability and possible shortcomings when it comes to these degradation events in biological fluids. Tracking protein stability in biological fluids typically requires purifying or labeling the protein of interest to circumvent matrix interference of biological fluids. Improved analytical methods are strongly needed to gain knowledge on in vivo protein aggregation and fragmentation. In vitro models can support the selection of lead candidates and accelerate the pre-clinical development of therapeutic proteins.