The Effect of Small Oligomeric Protein Aggregates on the Immunogenicity of Intravenous and Subcutaneous Administered Antibodies.

The role of aggregates in the immunogenicity of biologics is a major concern. A recent US FDA guidance on the issue suggests that a gap in knowledge exists regarding the type and size of aggregates involved in the immunogenicity of biologics. Furthermore, the guidance suggests that current techniques cannot capture the crucial stages of protein aggregation. Using a protein unfolding model developed earlier, we generated and classified aggregates of two therapeutic antibodies based on size and conformation. The immunogenic potential of these aggregates were then tested in a murine model. Our findings show that small native-like oligomeric aggregates (<100 nm) are more immunogenic toward the native protein than monomer and large non-native aggregates in the micron-size range, irrespective of route of administration [intravenous (i.v.) vs. subcutaneous (s.c.)]. Those smaller oligomeric aggregates represented 5%-20% of the total protein concentration in the test formulations. Furthermore, in vitro data suggest that TNF-α production by bone marrow-derived dendritic cells could serve as a predictive marker for increased immunogenic risk of aggregates after s.c. administration. The use of orthogonal techniques such as fluorescence anisotropy and quasielastic light scattering may be useful to detect these oligomeric aggregates.