Subunit mass analysis for monitoring multiple attributes of monoclonal antibodies

Abstract

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 coformulated 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.

The lysosomal endopeptidases Cathepsin D and L are selective and effective proteases for the middle‐down characterization of antibodies

“…These protein fragment precursors provide a corresponding sequence on which all fragment ions can and should be mapped. Combined with the MS1 intact information on the different fragments, particularly when determined at high resolving power, top-and middle-down proteomic approaches can be quite powerful [8][9][10][11][14][15][16][17][18][19][20][21].…”

An evaluation of instrument types for mass spectrometry-based multi-attribute analysis of biotherapeutics

“…Multi-attribute methods (MAM), based on proteolytic digestion and subsequent liquid chromatography-mass spectrometry (LC-MS) analysis of proteolytic peptides, are used to quantify a variety of quality attributes in protein therapeutics. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] These methods take advantage of the resolving power of a mass spectrometric (MS) detector and use the MS response of each peptide isoform for quantitation. Because of the specificity of these methods toward each clinically relevant quality attribute, they have gained extensive attention in the biopharmaceutical industry.…”

“…MS-based MAM has been performed on high-resolution mass spectrometers, [1][2][3][4][5][6][7][8][9][10][11] although some work has demonstrated the success of MAM on a low-resolution instrument. 12 Highresolution instruments, because of their high mass resolving power, are capable of resolving the analytes of interest from most interferences, and thereby providing superb analyte specificity.…”

Structure-Function Assessment and High-Throughput Quantification of Site-Specific Aspartate Isomerization in Monoclonal Antibody Using a Novel Analytical Tool Kit

“…18,[20][21][22][23][24] To ensure consistent efficacy and safety profiles of protein therapeutics during manufacturing and storage, the structure-function relationships and the degradation pathways of the protein need to be fully understood and appropriate control strategy must be established. For characterization, mass spectrometry (MS)-based multiattribute methods [25][26][27][28] and 2-dimensional liquid chromatography (2DLC) 29,30 with MS detection 31,32 may find more utility due to their efficiency in obtaining more information with a smaller set of analytical methods. However, orthogonal traditional approaches are still required in delivering the requisite data during characterization and release testing in quality control (QC) settings, which simultaneously deliver a more complete picture of the degradation pathways.…”