Mass Spectrometric Analysis of Intact Human Monoclonal Antibody Aggregates Fractionated by Size-Exclusion Chromatography

Publication Type:

Journal Article


Pharmaceutical Research, Volume 27, Number 10, pp. 2197-2204 (2010)



DOI Name (links to online publication)



hp-sec; igg1; monoclonal antibody; native esi-tof mass spectrometry; protein aggregation; multidomain protein; physical stability; structural biology; thermal-stress; dimer; immunoglobulin; therapeutics; conformation; formulation; assemblies


The aim of this study was to develop a method to characterize intact soluble monoclonal IgG1 antibody (IgG) oligomers by mass spectrometry.IgG aggregates (dimers, trimers, tetramers and high-molecular-weight oligomers) were created by subjecting an IgG formulation to several pH jumps. Protein oligomer fractions were isolated by high performance size exclusion chromatography (HP-SEC), dialyzed against ammonium acetate pH 6.0 (a mass spectrometry-compatible volatile buffer), and analyzed by native electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS).Monomeric and aggregated IgG fractions in the stressed IgG formulation were successfully isolated by HP-SEC. ESI-TOF MS analysis enabled us to determine the molecular weight of the monomeric IgG as well as the aggregates, including dimers, trimers and tetramers. HP-SEC separation and sample preparation proved to be necessary for good quality signal in ESI-TOF MS. Both the HP-SEC protocol and the ESI-TOF mass spectrometric technique were shown to leave the IgG oligomers largely intact.ESI-TOF MS is a useful tool complementary to HP-SEC to identify and characterize small oligomeric protein aggregates.