Two-Dimensional Capillary Zone Electrophoresis-Mass Spectrometry: Intact mAb Charge Variant Separation Followed by Peptide Level Analysis Using In-Capillary Digestion

Abstract

Characterization of charge heterogeneity is an essential pillar for pharmaceutical development and quality control of therapeutic monoclonal antibodies (mAbs). The highly selective and commonly applied capillary zone electrophoresis (CZE) method containing high amounts of ε-aminocaproic acid (EACA) provides a detailed and robust charge heterogeneity profile of intact mAb variants. Nevertheless, the exact location of protein modifications within these charge profiles remains ambiguous. Electrospray ionization mass spectrometry (ESI-MS) is a promising tool for this purpose; however, EACA is incompatible with electrospray. In this context, we present a two-dimensional CZE-CZE-MS system to combine efficient charge variant separation of intact mAbs with subsequent peptide analysis after in-capillary digestion of selected charge variants. The first dimension is based on a generic CZE(EACA) method in a fused silica capillary. In the second dimension, a neutral-coated capillary is used for in-capillary reduction and digestion with Tris(2-carboxyethyl)phosphine (TCEP) and pepsin, followed by CZE separation and MS/MS-characterization of the resulting peptides. The setup is demonstrated using stressed and nonstressed mAbs where peaks of basic, main, and acidic variants were transferred in a heart-cut fashion, digested, and characterized on the peptide level. Sequence coverages of more than 90% were obtained for heavy chain (HC) and light chain (LC) for four different mAbs, including low-abundance variants (<2% of the main peak). Frequently observed modifications (deamidation, oxidation, etc.) could be detected and localized. This study demonstrates a proof-of-concept for identification and localization of protein modifications from CZE charge heterogeneity profiles and, in this way, is expected to support the development and quality control testing of protein pharmaceuticals.

Recent developments in capillary and microchip electroseparations of peptides (2021–mid‐2023)

“…In‐capillary digestion of charge variants of unstressed and thermally stressed mAb by pepsin was monitored by 2D‐CE–ESI–MS setup described in Section 5.7. The peptide fragments were separated in 93 cm long 50 µm id capillary physically coated with PEO using 200 mM FA as BGE and 50% IPA, 0.2% FA as the SL with delivery flow rates of 3–8 µL/min [185], see Figure 8. The common PTMs of mAb, deamidation, oxidation, and others were detected and localized in the polypeptide chains.…”

“…A powerful 2D‐CZE‐CZE setup composed of two BFS capillaries connected via customized 8‐port valve and coupled with tandem ESI–MS detection was applied for the separation of charge variants of intact mAb in the first CZE dimension (in BGE composed of 380 mM e‐aminocaproic acid, 2 mM triethylenetetramine, 0.05% HPC, pH 5.7) and for separation complex mixture of peptide generated by online in capillary reduction of mAb with tris(2‐carboxyethyl)phosphine and digestion by pepsin in the second CZE dimension (in BGE consisted of 200 mM FA) [185]. Several common modifications as deamidation and oxidation were detected and localized in four different mAb molecules.…”

Multi‐dimensional technology – Recent advances and applications for biotherapeutic characterization

“…As the next evolution of mD‐CE, a novel two‐dimensional CZE‐CZE‐MS system was introduced [99]. This system effectively combines the separation of charge variants at the intact mAb level with rapid peptide analysis post‐in‐capillary digestion of specific charge variants.…”

“…(B) Analysis of non‐stressed and stressed (40°C for 4 weeks) trastuzumab samples using the CZE‐CZE‐MS set‐up with (Top) the 1D CZE‐UV signals, (Middle) the resulting electropherograms of selected peaks after peptide mapping in the 2 nd CZE dimension, and (Down) the specific ion traces corresponding to both non‐deamidated and deamidated species. Adapted [99], with permission.…”

Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications—Updated and completely revised edition

“…It allows unam-biguous peak identification and provides additional structural information about the analyzed protein species. Different approaches have been described, for example, online CIEF-MS [218,275,282,285], microchip CIEF-MS [207,217,286], CIEF-CZE-MS [287,288], and online iCIEF-MS [289][290][291][292]. In the case of direct online CIEF-MS, the catholyte and anolyte are additionally replaced with volatile compounds, for example, ammonia (ammonium hydroxide) and formic acid, and glycerol is used as dynamic coating and viscosity enhancing agent.…”