LAUSR

Protein aggregates are one of the several risk factors for undesired immunogenicity of biopharmaceuticals. However, it remains unclear which features determine whether aggregates will trigger an unwanted immune response. The aim of this study was to determine the effect of aggregates' size on their relative immunogenicity. A monoclonal murine IgG1 was stressed by exposure to low pH and elevated temperature followed by stirring to obtain aggregates widely differing in size. Aggregate fractions enriched in soluble oligomers, submicron size particles and micron size particles were isolated via centrifugation or size-exclusion chromatography and characterized physicochemically. The secondary and tertiary structures of aggregates were altered in a similar way for all the fractions, while no substantial chemical degradation was observed. Development of anti-drug antibodies was measured after subcutaneous administration of each enriched fraction to BALB/c mice. Among all tested fractions, the most immunogenic was the one highly enriched in submicron size particles (∼100-1000 nm). Fractions composed of micron size (>1-100 μm) particles or soluble oligomers (<100 nm) were not immunogenic under the dosing regimen studied in this work. These results show that aggregate size is an important factor for protein immunogenicity.