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Rationale: Multi‐Attribute Methods (MAMs) are appealing due to their ability to provide data on multiple molecular attributes from a single assay. If fully realized, such tests could reduce the number of assays required to support a product control strategy while providing equivalent or greater product understanding relative to the conventional approach. In doing so, MAMs have the potential to decrease development and manufacturing costs by reducing the number of tests in a release panel. Methods: In this work, we report a MAM which is based on subunit mass analysis. Results: The MAM assay is shown to be suitable for use as a combined method for identity testing, glycan profiling, and protein ratio determination for co‐formulated monoclonal antibody (mAb) drugs. This is achieved by taking advantage of the high mass accuracy and relative quantification capabilities of intact mass analysis using quadrupole time‐of‐flight mass spectrometry (Q‐TOF MS). Protein identification is achieved by comparing the measured masses of light chain (LC) and heavy chain (HC) mAbs against their theoretical values. Specificity is based on instrument mass accuracy. Glycan profiling and relative protein ratios are determined by the relative peak intensities of the protein HC glycoforms and LC glycoforms, respectively. Results for these relative quantifications agree well with those obtained by the conventional hydrophilic interaction liquid chromatography (HILIC) and reversed‐phase LC methods. Conclusions: The suitability of this MAM for use in a quality control setting is demonstrated through assessment specificity for mAb identity, and accuracy, precision, linearity and robustness for glycan profiling and ratio determination. Results from this study indicate that a MAM with subunit mass analysis has the potential to replace three conventional methods widely used for mAb release testing including identification assay, glycosylation profiling, and ratio determination for co‐formulated mAbs. [ABSTRACT FROM AUTHOR]

In top-down proteomics, intact gaseous proteins are fragmented in a mass spectrometer by, e.g., electron capture dissociation (ECD) to obtain structural information. By far, most top-down approaches involve dissociation of protein cations. However, in electrospray ionization of phosphoproteins, the high acidity of phosphate may contribute to the formation of intramolecular hydrogen bonds or salt bridges, which influence subsequent fragmentation behavior. Other acidic proteins or proteins with regions containing multiple acidic residues may also be affected similarly. Negative ion mode, on the other hand, may enhance deprotonation and unfolding of multiply phosphorylated or highly acidic protein regions. Here, activated ion electron detachment dissociation (AI-EDD) and negative ion infrared multiphoton dissociation (IRMPD) were employed to investigate the fragmentation of intact proteins, including multiply phosphorylated β-casein, calmodulin, and glycosylated ribonuclease B. Compared to AI-ECD and positive ion IRMPD, AI-EDD and negative ion IRMPD provide complementary protein sequence information, particularly in regions with high acidity, including the multiply phosphorylated region of β-casein. [ABSTRACT FROM AUTHOR]