Monoclonal antibodies (mAbs) are highly complex glycoproteins that present a wide range of microheterogeneities that requires multiple analytical methods for full structure assessment and quality control. Capillary zone electrophoresis-mass spectrometry (CZE-MS) couplings, especially by electrospray ionization (ESI), appear to be really attractive methods for the characterization of biological samples. However, due to the presence of non- or medium volatile salts in the background electrolyte (BGE), online CZE-ESI-MS coupling is difficult to implement for mAbs isoforms separation. Here, we report an original strategy to perform off-line CZE-ESI-MS using CZE-UV/fraction collection technology to perform CZE separation, followed by ESI-MS infusion of the different fractions using the capillary electrophoresis-electrospray ionization (CESI) interface as the nanoESI infusion platform. As the aim is to conserve electrophoretic resolution and complete compatibility with ESI-MS without sample treatment, hydroxypropylcellulose (HPC) coated capillary was used to prevent analyte adsorption and asymmetric CZE conditions involving different BGE at both ends of the capillary have been developed. The efficiency of our strategy was validated with the separation of Cetuximab charge variant by the middle-up approach. Molecular weights were measured for six charge variants detected in the CZE separation of Cetuximab subunits. The first three peaks correspond to Fc/2 variants with electrophoretic resolution up to 2.10, and the last three peaks correspond to F(ab')2 variants with average electrophoretic resolution of 1.05. Two Fc/2 C-terminal lysine variants were identified and separated. Moreover, separation of Fc/2 fragments allowed the glycoprofiling of the variants with the characterization of 7 different glycoforms. Regarding the F(ab')2 domain, 8 glycoforms were detected and separated in three different peaks following the presence of N-glycolyl neuraminic acid residues in some glycan structures. This work highlights the potential of CZE technology to perform separation of mAbs especially when they carry sialic acid carbohydrates.

Monoclonal antibodies (mAbs) are highly complex proteins that display a wide range of microheterogeneity that requires multiple analytical methods for full structure assessment and quality control. As a consequence, the characterization of mAbs on different levels is particularly product - and time - consuming. This work presents the characterization of trastuzumab sequence using sheathless capillary electrophoresis (referred as CESI) – tandem mass spectrometry (CESI-MS/MS). Using this bottom-up proteomic-like approach, CESI-MS/MS provided 100% sequence coverage for both heavy and light chain via peptide fragment fingerprinting (PFF) identification. The result was accomplished in a single shot, corresponding to the analysis of 100 fmoles of digest. The same analysis also enabled precise characterization of the post-translational hot spots of trastuzumab, used as a representative widely marketed therapeutic mAb, including the structural confirmation of the five major N-glycoforms.

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.