Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis – Mass spectrometry

Abstract

Significant growth of biopharmaceuticals requires powerful analytical methods to better understand their structure by establishing a complete characterization. To this end, a combination of bottom-up, middle-up and intact molecule levels with a capillary electrophoresis-mass spectrometry coupling has been performed to have a comprehensive picture of monoclonal antibodies. In this study, 7 worldwide health authorities approved mAbs have been analyzed to get information about their charge heterogeneity, the identification of post translational modifications (PTMs), their location and relative quantitation. Intact mAbs isoforms have been partially separated in less than 12 minutes and enabled to have a global illustration of mAbs heterogeneity and high masses PTMs characterization notably major N-glycosylation forms. Particularly, 2X-glycosylated and 1X-glycosylated forms have been partially separated. To deepen characterize PTMs carried by the backbone structure, advanced investigations at a middle-up level have been performed. Limited IdeS proteolysis allowed to study independently Fc/2 and F(ab)'2 fragments. Following the same separation conditions, isoforms of these fragments have been separated and data interpretation allowed to disclose additional PTMs as K-clip, oxidations or deamidations. A second intermediate level has been examined by adding a reduction step to establish a more precise assessment of PTMs and isoforms from the F(ab)'2 fragment. This reduction step released the light chains from the Fd fragment to get only 25 kDa fragments to analyze. CE-ESI-MS coupling allowed to get more information particularly about low masses PTMs. The precise location and relative quantitation of each PTM has been investigated at the peptidic level induced by a tryptic digestion of the studied mAbs. The concordance of the results shows the efficiency of the CE-ESI-MS coupling to characterize mAbs and highlight the need of the multi-level combination to get a comprehensive characterization of biotherapeutics.

Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches

“…The off-line sample preparation comprising a digestion and reduction steps can be performed within one hour, and requires a low amount of material (104). The 25 kDa fragments can then be easily separated by LC (107)(108)(109) or CE (110)(111)(112)(113) and simultaneously identified by HRMS instruments such as QTOFs and Orbitrap analyzers that can resolve the isotopic pattern of 25kDa proteins with a mass accuracy around 5 ppm. The middledown approach, which refers to MS/MS-based experiments, can also be performed for sequencing these fragments (114).…”

“…Several studies highlighted the advantages of combining the analysis of antibodies at multiple levels, i.e. intact, reduced, middle-up, and peptide level, to allow a fast and extensive characterization (111,(131)(132)(133). For instance, Giorgetti et al reported the combination of intact, middle-up, and bottom-up analysis using a CE-MS instrument for the analysis of 7 FDA-approved mAbs(111) with: i) the identification of major N-glycoforms at the intact level; ii) additional PTMs as K-clip, oxidations or deamidations observed at the middle-up level; and iii) a precise location and relative quantitation of each PTM at the bottom-up level.…”

Applications of capillary electrophoresis for biopharmaceutical product characterization

“…The applicability of CE-MS in PTM analysis was demonstrated with seven commercial therapeutic antibodies [62]. The method was used to identify glycoforms and other PTMs, especially low mass PTMs, using intact, middle-up, and bottom-up approaches.…”

“…The presence of major glycoforms, that is, 2xglycosylated and 1x-glycosylated isoforms, in seven commercially available mAbs was recently analyzed in less than 12 min using CE-MS of intact and IdeS digested forms [62]. Glycosylation analysis has also been reported via relative quantification of N-glycans at the glycopeptide level [68].…”

Immunoaffinity Capillary Electrophoresis in the Era of Proteoforms, Liquid Biopsy and Preventive Medicine: A Potential Impact in the Diagnosis and Monitoring of Disease Progression

“…Proteomics should ideally be the driver for improving diagnostics and will serve as a primary method for detection of molecular changes indicative of disease states. Numerous laboratories have reported a plethora of assays using capillary electrophoresis in conventional format and in microfluidic format for detection and quantification of several proteoforms [254][255][256][257][258][259][260][261][262][263][264][265][266][267]. However, in order for proteoforms to reach their full clinical potential, it is necessary to be able to accurately detect and quantify near all proteoforms in biological fluids using advanced and sophisticated analytical instrumentation, such as IACE.…”