Monoclonal antibodies (mAbs) and their related products as antibody-drug-conjugates (ADCs) or biosimilars represent a constantly growing class of molecules therapeutic proteins used as treatment against numerous diseases. These compounds can undergo several modifications which could alter the efficiency of treatments. In this context, several analytical methods were designed to deliver a comprehensive structural characterization and guarantee the quality of biotherapeutics. Capillary electrophoresis (CE) is considered today as a major technique for the analysis of biotherapeutics due to benefic characteristics as high resolution separation and miniaturized format. Different CE modes have been developed to characterize mAbs at different levels such as capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF), and capillary zone electrophoresis (CZE). Recent developments in CE-mass spectrometry (MS) coupling assessed this technology as a promising tool to obtain high level structural characterization of biopharmaceuticals. Moreover, upcoming techniques such as 2D CE-MS and microfluidic systems are now emerging to offer new possibilities beyond actual limits. This review will be dedicated to discuss the state-of-the-art CE-based methods for the characterization of mAbs and ADCs in the period 2016-2018.
A B S T R A C TCharacterization of therapeutic proteins represents a major challenge for analytical sciences due to their heterogeneity caused by post-translational modifications (PTM). Among these PTM, glycosylation which is possibly the most prominent, require comprehensive identification because of their major influence on protein structure and effector functions of monoclonal antibodies (mAbs). As a consequence, glycosylation profiling must be deeply characterized. For this application, several analytical methods such as separation-based or MS-based methods, were evaluated. However, no CE-ESI-MS approach has been assessed and validated. Here, we illustrate how the use of CE-ESI-MS method permits the comprehensive characterization of mAbs N-glycosylation at the glycopeptide level to perform relative quantitation of N-glycan species. Validation of the CE-ESI-MS method in terms of robustness and reproducibility was demonstrated through the relative quantitation of glycosylation profiles for ten different mAbs produced in different cell lines. Glycosylation patterns obtained for each mAbs were compared to Hydrophilic Interaction Chromatography of 2-aminobenzamide labelled glycans with fluorescence detector (HILIC-FD) analysis considered as a reference method. Very similar glycoprofiling were obtained with the CE-ESI-MS and HILIC-FD demonstrating the attractiveness of CE-ESI-MS method to characterize and quantify the glycosylation heterogeneity of a wide range of therapeutic mAbs with high accuracy and precision.
Capillary zone electrophoresis-mass
spectrometry (CE-MS) is a mature
analytical tool for the efficient profiling of (highly) polar and
ionizable compounds. However, the use of CE-MS in comparison to other
separation techniques remains underrepresented in metabolomics, as
this analytical approach is still perceived as technically challenging
and less reproducible, notably for migration time. The latter is key
for a reliable comparison of metabolic profiles and for unknown biomarker
identification that is complementary to high resolution MS/MS. In
this work, we present the results of a Metabo-ring trial involving
16 CE-MS platforms among 13 different laboratories spanning two continents.
The goal was to assess the reproducibility and identification capability
of CE-MS by employing effective electrophoretic mobility (μ
eff
) as the key parameter in comparison to the relative migration
time (RMT) approach. For this purpose, a representative cationic metabolite
mixture in water, pretreated human plasma, and urine samples spiked
with the same metabolite mixture were used and distributed for analysis
by all laboratories. The μ
eff
was determined for
all metabolites spiked into each sample. The background electrolyte
(BGE) was prepared and employed by each participating lab following
the same protocol. All other parameters (capillary, interface, injection
volume, voltage ramp, temperature, capillary conditioning, and rinsing
procedure, etc.) were left to the discretion of the contributing laboratories.
The results revealed that the reproducibility of the μ
eff
for 20 out of the 21 model compounds was below 3.1% vs 10.9% for
RMT, regardless of the huge heterogeneity in experimental conditions
and platforms across the 13 laboratories. Overall, this Metabo-ring
trial demonstrated that CE-MS is a viable and reproducible approach
for metabolomics.
Since last decade there is a growing interest of RNA modifications analysis. High performance liquid chromatography-tandem mass spectrometry coupling (HPLC-MS/MS) is classically used to characterize post-transcriptional modifications of ribonucleic acids (RNAs). Here we proposed a novel and simple workflow based on capillary zone electrophoresis-tandem mass spectrometry (CE-MS/MS), in positive mode, to characterize RNA modifications at nucleoside and oligonucleotide levels. A first total digestion of purified RNA, prior CE-MS/MS analysis, enables to identify nucleoside modifications. Then, using a bottom-up approach, sequencing of RNAs and mapping of modifications were performed. Sequence coverages from 68% to 97% were obtained for four tRNAs. Furthermore, unambiguous identification and mapping of several modifications were achieved.
As RNA post-transcriptional modifications are of growing interest, several methods were developed for their characterization. One of them established for their identification, at the nucleosidic level, is the hyphenation of separation methods, such as liquid chromatography or capillary electrophoresis, to tandem mass spectrometry. However, to our knowledge, no software is yet available for the untargeted identification of RNA post-transcriptional modifications from MS/MS data-dependent acquisitions. Thus, very long and tedious manual data interpretations are required. To meet the need of easier and faster data interpretation, a new user-friendly search engine, called Nucleos'ID, was developed for CE-MS/ MS and LC−MS/MS users. Performances of this new software were evaluated on CE-MS/MS data from nucleoside analyses of already well-described Saccharomyces cerevisiae transfer RNA and Bos taurus total tRNA extract. All samples showed great true positive, true negative, and false discovery rates considering the database size containing all modified and unmodified nucleosides referenced in the literature. The true positive and true negative rates obtained were above 0.94, while the false discovery rates were between 0.09 and 0.17. To increase the level of sample complexity, untargeted identification of several RNA modifications from Pseudomonas aeruginosa 70S ribosome was achieved by the Nucleos'ID search following CE-MS/MS analysis.
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