Quantification of Monoclonal Antibodies in Pharmaceutical Matrices by Middle-Up Capillary Zone Electrophoresis-Mass Spectrometry.

Strategy for selecting separation solutions in capillary electrophoresis–mass spectrometry

Identification of a monoclonal antibody clipping variant by cross-validation using capillary electrophoresis – sodium dodecyl sulfate, capillary zone electrophoresis – mass spectrometry and capillary isoelectric focusing – mass spectrometry

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 (μeff) 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 μeff 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 μeff 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.

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.

Identification of 3-hydroxyaspartate with two chiral centers by capillary electrophoresis-mass spectrometry and vibrational circular dichroism independent of single enantiomer standard

Characterization of cetuximab Fc/2 dimers by off-line CZE-MS

Analysis of tetramine in sea snails by capillary electrophoresis-tandem mass spectrometry

Capillary electrophoresis-mass spectrometry often lacks sufficient limits of detection for trace substances in the environment due to its low loadability. To overcome this problem, we conducted a feasibility study for column-coupling isotachophoresis to capillary electrophoresis-mass spectrometry. The first dimension isotachophoresis preconcentrated the analytes. The column-coupling of both dimensions was achieved by a hybrid capillary microfluidic chip setup. Reliable analyte transfer by voltage switching was enabled by an in-chip capacitively coupled contactless conductivity detector placed around the channel of the common section between two T-shaped crossings in the chip connecting both dimensions. This eliminated the need to calculate the moment of analyte transfer. A commercial capillary electrophoresis-mass spectrometry instrument with easily installable adaptations operated the setup. Prior to coupling isotachophoresis with capillary zone electrophoresis-mass spectrometry, both dimensions were optimized individually by simulations and verified experimentally. Both dimensions were able to stack/separate all degradation products of glyphosate, the most important herbicide applied worldwide. The first dimension isotachophoresis also removed phosphate, which is a critical matrix component in many environmental samples. Enrichment and separation of glyphosate and its main degradation product aminomethylphosphonic acid by the two-dimensional setup provided an excellent limit of detection of 150pM (25ng/L) for glyphosate.

Investigating native capillary zone electrophoresis-mass spectrometry on a high-end quadrupole-time-of-flight mass spectrometer for the characterization of monoclonal antibodies

High-resolution capillary zone electrophoresis - mass spectrometry (CZE-MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle-down and intact CZE-MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post-translational modifications (PTMs) and glycosylation structures. Middle-down and intact CZE separations were performed in an acidified methanol-water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle-down analysis of the IdeS-digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X-deamidated, 1X-deamidated, and non-deamidated forms at baseline resolution. In the course of the middle-down CZE-MS analysis, separation of glycoforms of the FC /2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE-MS2 . Incorporation of TCEP-HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE-MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X-glycosylated, 1X-glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE-MS represents a complementary approach to the more conventional liquid-chromatography - mass spectrometry-based approaches.

Derivatization reagent-assisted enantioseparation of 3-hydroxyaspartate with two chiral centers in rat cerebrospinal fluid by capillary electrophoresis-mass spectrometry

The aspiration of the multi-attribute method (MAM) is to utilize a single mass spectrometry-based method that can measure multiple attributes simultaneously, thus enabling data-driven decisions more quickly and efficiently. However, challenges associated with identifying and quantitating critical quality attributes such as asparagine deamidation and isoaspartic acid using conventional ultrahigh-pressure liquid chromatography (UHPLC) coupled to mass spectrometry have necessitated long gradients to ensure sufficient separation for quantitation. Microfluidic chip-based capillary zone electrophoresis mass spectrometry (CZE-MS) shows potential to enable rapid charge-based separation of peptide mixtures, and this approach was evaluated using multipeptide mixtures of synthetic peptides as well as digested protein therapeutics. In these experiments, repeatability, linearity, and peak-to-peak resolution of several peptide families containing asparagine deamidation and/or isoaspartic acid were demonstrated. In addition, a comparison of peptide map results acquired with both UHPLC-MS and CZE-MS for two enzymatically digested biological therapeutics showed comparable sequence coverage and quantitation results between the two approaches. As MAM becomes increasingly utilized for analysis of biological therapeutics, MS instrument demand will rapidly increase, resulting in a bottleneck. A CZE-based separation shows potential to alleviate this bottleneck by drastically increasing MAM throughput while providing results comparable to those acquired using conventional UHPLC separations.

An evaluation of capillary zone electrophoresis-mass spectrometry (CZE-MS) as an analytical methodology for the separation and characterization of complex glycopeptides and nonglycopeptide structures has been performed. The evaluation employed endoproteinase V8 digested recombinant human erythropoietin (rHuEPO) that was further fractionated by reverse phase chromatography. The peptides were subjected to sequence analysis and evaluated by capillary electrophoresis, with or without mass detection, for peptide purity. The peptide mass determined from the sequence was then compared to the mass obtained from CZE-MS. Glycosylation sites and carbohydrate branch patterns were easily determined, site specific microheterogeneity (either O-acetylation of N-acetylneuraminic acids or lactosamine extensions of the carbohydrate chain length) was assessed directly, glycosylation site occupancy was evaluated qualitatively, and nonglycopeptides were resolved and analyzed on-line with ease. Incomplete peptide digestion products were detected and identified by CZE-MS. Protein sequence coverage by CZE-MS was 98.2 percent complete from a single map. Off-line evaluation of peptide purity by CZE greatly aided the interpretation of multiple sequence analysis and, in validating that, the CZE-MS was detecting all peptides present. All off-line CZE and on-line CZE-MS experiments employed a capillary that was dynamically coated with Polybrene in the presence of polyethylene glycol; separations were conducted in 0.67 M formic acid.

Production of site-specific cysteine-engineered antibody-drug conjugates (ADCs) in mammalian cells may produce developability challenges, fragments, and heterogenous molecules, leading to potential product critical quality attributes in later development stages. Liquid phase chromatography with mass spectrometry (LC-MS) is widely used to evaluate antibody impurities and drug-to-antibody ratio, but faces challenges in analysis of fragment product variants of cysteine-engineered ADCs and oligonucleotide-to-antibody ratio (OAR) species of antibody-oligonucleotide conjugates (AOCs). Here, for the first time, we report novel capillary zone electrophoresis (CZE)-MS approaches to address the challenges above. CZE analysis of six ADCs made with different parent monoclonal antibodies (mAbs) and small molecule drug-linker payloads revealed that various fragment impurities, such as half mAbs with one/two drugs, light chains with one/two drugs, light chains with C-terminal cysteine truncation, heavy chain clippings, were well resolved from the main species. However, most of these fragments were coeluted or had signal suppression during LC-MS analysis. Furthermore, the method was optimized on both ionization and separation aspects to enable the characterization of two AOCs. The method successfully achieved baseline separation and accurate quantification of their OAR species, which were also highly challenging using conventional LC-MS methods. Finally, we compared the migration time and CZE separation profiles among ADCs and their parent mAbs, and found that properties of mAbs and linker payloads significantly influenced the separation of product variants by altering their size or charge. Our study showcases the good performance and broad applicability of CZE-MS techniques for monitoring the heterogeneity of cysteine-engineered ADCs and AOCs.

Four methods were compared for analysis of host-cell protein (HCP) impurities in a recombinant mAb. First, CZE-MS/MS was used to analyze the digest of an HCP sample following extraction of the mAb with proteins A and L affinity columns; 220 protein groups and 976 peptides were identified from the depleted HCP digest. Second, a nanoACQUITY UltraPerformance LCH system was also used to analyze the depleted HCP digest; 34 protein groups and 53 peptides from 50 ng of the depleted HCP digest and 290 protein groups and 1011 peptides were identified from 1 μg of the depleted HCP digest. Third, 185 protein groups and 709 peptides were identified by CZE-MS/MS from the HCP digest without depletion. Fourth, a strong cation exchange SPE was coupled to CZE-ESI-MS/MS using online pH gradient elution for analysis of the HCP digest without depletion. A series of five pH bumps were applied to elute peptides from the strong cation exchange monolith followed by analysis using CZE coupled to a Q Exactive HF mass spectrometer; 230 protein groups and 796 peptides were identified from the HCP digest without depletion.