Unbiased in-depth characterization of CEX fractions from a stressed monoclonal antibody by mass spectrometry

Abstract

Characterization of charge-based variants by mass spectrometry (MS) is required for the analytical development of a new biologic entity and its marketing approval by health authorities. However, standard peak-based data analysis approaches are time-consuming and biased toward the detection, identification, and quantification of main variants only. The aim of this study was to characterize in-depth acidic and basic species of a stressed IgG1 monoclonal antibody using comprehensive and unbiased MS data evaluation tools. Fractions collected from cation ion exchange (CEX) chromatography were analyzed as intact, after reduction of disulfide bridges, and after proteolytic cleavage using Lys-C. Data of both intact and reduced samples were evaluated consistently using a time-resolved deconvolution algorithm. Peptide mapping data were processed simultaneously, quantified and compared in a systematic manner for all MS signals and fractions. Differences observed between the fractions were then further characterized and assigned. Time-resolved deconvolution enhanced pattern visualization and data interpretation of main and minor modifications in 3-dimensional maps across CEX fractions. Relative quantification of all MS signals across CEX fractions before peptide assignment enabled the detection of fraction-specific chemical modifications at abundances below 1%. Acidic fractions were shown to be heterogeneous, containing antibody fragments, glycated as well as deamidated forms of the heavy and light chains. In contrast, the basic fractions contained mainly modifications of the C-terminus and pyroglutamate formation at the N-terminus of the heavy chain. Systematic data evaluation was performed to investigate multiple data sets and comprehensively extract main and minor differences between each CEX fraction in an unbiased manner.

Identification of multiple serine to asparagine sequence variation sites in an intended copy product of LUCENTIS® by mass spectrometry

“…2B ). 26 In brief, this process enables the deconvolution of each 1s MS scan in parallel for all samples processed altogether. Such display retains for each species the connection between deconvoluted mass and respective retention time.…”

“…Robust sample comparison was enabled using in parallel time-resolved deconvolution of each acquired MS scan in an unbiased manner. 26 This feature improved the identification of product-related variants by pattern recognition. Additionally, statistical head-to-head comparison of peptide maps using searches for absent and present signals enabled the identification of differences in RAZUMAB primary sequence at multiple sites (+ 27.01 Da as monoisotopic mass shift), which were confirmed as serine to asparagine substitution.…”

Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry

“…These experimental procedures usually involve peak collection followed by buffer exchange to allow further analysis, which can be time consuming and costly. 15 More importantly, these procedures are a possible source for sample preparation-induced modifications.…”

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

“…[22][23][24] Upon the separation of charge variants by IEX, current strategies to determine the effects of modifications on specific charge variant peak involve isolating the peak of interest followed by various mass spectrometric analyses, such as intact mass analysis, peptide mapping, and glycan analysis. 10,25 In addition to being limited by time and resources, this two-step approach may overlook the minor species that do not exhibit distinctive UV peaks and introduce artifacts because of the lengthy sample preparation processes. 26 Therefore, the ability to directly couple IEX to high-resolution mass spectrometry (MS) is highly desirable to enable sensitive MS detection, which can significantly improve efficiency for charge heterogeneity characterization.…”

“…[40][41][42][43][44] Additionally, CEX has greater resolving power as compared to RP LC and allows complete separation of intact antibody molecules even with one charged modification or a modification leading to partial net charge change. 7,10,23,29,35 With a few exceptions, 45 RP separation of protein charge variants with one modification is typically relatively poor, 46,47 but RP LC-MS is sensitive and easily amenable to top-down analysis. 43,[48][49][50][51] This is because proteins elute in the RP mobile phase in an unfolded and highly charged state, 52 which enables ionization prior to entering the mass analyzer.…”