Full Antibody Primary Structure and Microvariant Characterization in a Single Injection Using Transient Isotachophoresis and Sheathless Capillary Electrophoresis–Tandem Mass Spectrometry

Abstract

Here we report the complete characterization of the primary structure of a multimeric glycoprotein in a single analysis by capillary electrophoresis (CE) coupled to mass spectrometry (MS). CE was coupled to electrospray ionization tandem MS by means of a sheathless interface. Transient isotachophoresis (t-ITP) was introduced in this work as an electrokinetically based preconcentration technique, allowing injection of up to 25% of the total capillary volume. Characterization was based on an adapted bottom-up proteomic strategy. Using trypsin as the sole proteolytic enzyme and data from a single injection per considered protein, 100% of the amino acid sequences of four different monoclonal antibodies could be achieved. Furthermore, illustrating the effectiveness and overall capabilities of the technique, the results were possible through identification of peptides without tryptic miscleavages or posttranslational modifications, demonstrating the potency of the technique. In addition to full sequence coverages, posttranslational modifications (PTMs) were simultaneously identified, further demonstrating the capacity of this strategy to structurally characterize glycosylations as well as faint modifications such as asparagine deamidation or aspartic acid isomerization. Together with the exquisite detection sensitivity observed, the contributions of both the CE separation mechanism and selectivity were essential to the result of the characterization with regard to that achieved with conventional MS strategies. The quality of the results indicates that recent improvements in interfacing CE-MS coupling, leading to a considerably improved sensitivity, allows characterization of the primary structure of proteins in a robust and faster manner. Taken together, these results open new research avenues for characterization of proteins through MS.

Cutting‐edge mass spectrometry characterization of originator, biosimilar and biobetter antibodies

“…CESI‐MS is capable of analyzing simultaneously very small (<300 Da) and very large peptides (>8000 Da) under the same experimental conditions achieving 100% sequence coverage within one single run and one enzymatic digest. High ionization efficiencies provided by CESI and highly sensitive MS allowed in‐depth analysis of glycosylations with relative abundance varying more than 3 orders of magnitude . As a further evaluation, we have used CESI‐MS/MS to compare trypsin maps of trastuzumab and trastuzumab‐B and cetuximab and cetuximab‐B .…”

Monoclonal antibody N-glycosylation profiling using capillary electrophoresis – Mass spectrometry: Assessment and method validation

“…This property may be important, especially in case of more complex glycoproteins or for mAbs having several glycosylation sites. Moreover, it must be mentioned that our CE-ESI-MS methodology is not restricted to the sole purpose of glycopeptides characterization and quantitation, but it can also be employed for the overall characterization of mAbs, including primary structure assessment with complete sequence coverage as well as identification and quantification of a large number of PTMs, all performed within a unique single analysis [27]. In our opinion, this work proves that CE-ESI-MS could be a viable alternative to LC-ESI-MS for glycosylation profiling and should be considered as an innovative approaches in MS-based proteomics applied to mAbs characterization.…”

“…Despite significant instrumental development to achieve LIF and MS dual detection, their strategies allowed to perform quantitative analysis provided by the on-line LIF trace and to increase confidence by providing accurate mass information [26]. More recently, Gahoual et al reported the development of CE-ESI-MS technique for the characterization of the primary structure of mAbs performed in a single injection [27]. Based on a bottom-up approach, they highlighted the benefits of using electrophoretic separation in complement to chromatographic separation, which is conventionally applied in this type of study.…”

Glycoform Separation and Characterization of Cetuximab Variants by Middle-up Off-Line Capillary Zone Electrophoresis-UV/Electrospray Ionization-MS

“…11 More recently, Gahoual et al characterized 15 glycopeptides using a bottom-up approach by CESI-MS/MS. 13 In our work, we could identify a total of 15 glycans divided between 7 for the Fc/2 N-glycosylation and 8 for the F(ab′) 2 N-glycosylation (Tables S-1 and S-2, Supporting Information). Regarding the Fc/2 domain, branched glycan structures are primarily composed of fucose, Nacetylglucosamine, and mannose but can differ in the number of terminal galactose, mannose, or N-acetylglucosamine residues.…”