Structural properties of monoclonal antibody aggregates induced by freeze–thawing and thermal stress

Abstract

Aggregation of monoclonal antibodies can be induced by freeze–thawing and elevated temperature, typical stress factors during development, production and storage. Our aim was to characterize structural properties of aggregates formed after freeze–thawing and thermal stressing of humanized monoclonal IgG 1 antibody (IgG). Formulations with 1.0 mg/ml IgG in 100 mM phosphate pH 7.2 were subjected to freeze–thawing and heating and characterized by spectroscopic techniques (UV-absorption, CD, ATR-FTIR and fluorescence), light obscuration, dynamic light scattering, SDS-PAGE, AF4 with UV and MALLS detection, and HP-SEC with UV and online fluorescent dye detection. Thermal stress led to an increased formation of dimers and soluble oligomers (HP-SEC, AF4). Aggregates smaller than 30 nm were measured (DLS), next to slightly elevated particle levels in the μm range (light obscuration). Aggregates created by heating were in part covalently linked (SDS-PAGE) and made up of conformationally perturbed monomers (CD, ATR-FTIR, extrinsic dye fluorescence). Aggregation after freeze–thawing was manifested primarily in particle formation in the μm range. These aggregates were noncovalently linked (SDS-PAGE) and composed of native-like monomers, as obvious from CD, ATR-FTIR and extrinsic dye fluorescence spectroscopy. In conclusion, the complementary methods used in this study revealed that heating and freeze–thawing induced aggregates differ significantly in their physico-chemical characteristics.