title: 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 1.0mg/ml IgG in 100mM 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 30nm were measured (DLS), next to slightly elevated particle levels in the mum 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 mum 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.
authors.affiliation: Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, Leiden University, 2300 RA Leiden, The Netherlands. ahawe@chem.leidenuniv.nl
authors.forename: Andrea
authors.initials: A
authors.lastname: Hawe
authors.forename: Julia Christina
authors.initials: JC
authors.lastname: Kasper
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Friess
authors.forename: Wim
authors.initials: W
authors.lastname: Jiskoot
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005615:Freezing; D006358:Hot Temperature; D006801:Humans; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D000911:Antibodies, Monoclonal