Rapid and multi-level characterization of trastuzumab using sheathless capillary electrophoresis-tandem mass spectrometry

Abstract

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.

Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry

“…In a previous work, we demonstrated the capacity to obtain the complete sequence coverage in a single analysis using CESI-MS/MS. 23 It is important to note, however, that significant improvement of the methodology enabled us to obtain the complete AA sequence characterization exclusively through the identification of peptides, without miscleavages nor posttranslational modifications, as emphasized in Figure 2. Trastuzumab-B was characterized using the same methodology.…”

“…Tryptic peptides (without miscleavages or PTMs except cys carbamidomethylation) were determined theoretically from the mAbs' amino acid sequences available through literature. 23,47 Additional peptides were identified using Mascot search engine provided by Matrix science; tryptic cleavage rules were applied. Carbamidomethylation of cysteine (C57.02 Da), N-deamidation of aspartic/isoaspartic acid (C0.985 Da) or succinimide intermediate (¡17.03 Da), methionine oxidation (C15.99 Da) and N-terminal glutamic acid cyclization (-17.02 Da) were selected as variable modifications.…”

“…[16][17][18][19][20][21][22] Recently, we described a method using capillary zone electrophoresis (CZE) coupled to tandem MS with a sheathless interface to perform the simultaneous characterization of several aspects of a protein in one injection, including AA sequence and posttranslational modifications (PTMs). 23 The sheathless CE-ESI-MS interface, referred to as CESI-MS, has been developed based on an original design proposed by Moini et al 24 Detailed description of this interface has been given by Haselberg et al 25 The CESI system has been used to perform successful CE-ESI-MS experiments in proteomics, 26,27 metabolomics, 28 intact proteins, 25 therapeutic small molecules 29 and as an infusion platform, 30 and it demonstrated a drastically increased sensitivity compared to sheath-liquid CE-MS interfaces because of its low operating flow rates below 100 nL/min. In the present work, we developed a transient isotachophoresis CE-ESI-MS/MS methodology, with the CESI-MS system, to first obtain the characterization of several mAbs.…”

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

“…It only represents 2-5% of the total mass of the protein but it is subjected to extensive studies due to its significant influence on effector functions of mAbs, especially antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) [5][6][7][8]. As a consequence, the mAbs glycosylation profile is considered as a critical quality attribute (CQA) and must be thoroughly analyzed [9][10][11][12][13]. The complexity and heterogeneity of the glycosylation pattern is mainly due to mAbs production in living expression systems [14][15][16] and requires a number of orthogonal analytical techniques to be fully characterized.…”

Differential detection of Rhizoma coptidis by capillary electrophoresis electrospray ionization mass spectrometry with a nanospray interface

“…A sheathless CE-MS assay with a high sensitivity porous sprayer (HSPS) developed by Moini [27] was published worldwide from its mechanisms [28] to applications in amino acid analysis [29,30], glycan analysis [31,32], metabolomics [33][34][35][36], proteomics [26,[37][38][39], and protein interactions [40,41]. However, there was still less discussion in its detailed but important manipulations [26,42] and applications in analyzing Chinese medicine.…”