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Aggregates and fragments are common product-related impurities in biopharmaceuticals that have impact on efficacy, safety, and stability. Identification of these low-level impurities may involve labor-intensive chromatographic fraction collection and follow-up experiments. In addition, a significant part of the total protein aggregates can be caused by non-covalent molecular interactions, which are dissociable in denaturing buffers, and cannot be characterized by conventional mass spectrometry (MS). With the use of ammonium acetate buffer, size-exclusion chromatography (SEC) can be coupled on-line to electrospray ionization MS for the characterization of size variants of therapeutical monoclonal antibodies (mAbs). A quadrupole time-of-flight (QTOF) MS system was employed, and the MS method was optimized to achieve favorable sensitivity for high-mass detection, while maintaining the structural integrity of the aggregates. As a result, high quality native MS spectra could be acquired for drug substance samples containing as low as 0.4% of these species, with the molecular masses of all relevant size variants obtained using 10-min SEC-MS runs. It is expected that this method will have significant potential to be implemented in routine analysis of mAbs for the characterization of the aggregation mechanisms and degradation pathways. [ABSTRACT FROM AUTHOR]