Two-dimensional capillary zone electrophoresis-mass spectrometry for the characterization of intact monoclonal antibody charge variants, including deamidation products.

Capillary zone electrophoresis (CZE) is a powerful tool that is progressively being applied for the separation of monoclonal antibody (mAb) charge variants. Mass spectrometry (MS) is the desired detection method concerning identification of mAb variants. In biopharmaceutical applications, there exist optimized and validated electrolyte systems for mAb variant quantification. However, these electrolytes interfere greatly with the electrospray ionization (ESI) process. Here, a heart-cut CZE-CZE-MS setup with an implemented mechanical four-port valve interface was developed that used a generic ε-aminocaproic acid based background electrolyte in the first dimension and acetic acid in the second dimension. Interference-free, highly precise mass data (deviation less than 1 Da) of charge variants of trastuzumab, acting as model mAb system, were achieved. The mass accuracy obtained (low parts per million range) is discussed regarding both measured and calculated masses. Deamidation was detected for the intact model antibody, and related mass differences were significantly confirmed on the deglycosylated level. The CZE-CZE-MS setup is expected to be applicable to a variety of antibodies and electrolyte systems. Thus, it has the potential to become a compelling tool for MS characterization of antibody variants separated in ESI-interfering electrolytes. Graphical Abstract Two-dimensional capillary zone electrophoresis mass spectrometry for the characterization of intact monoclonal antibody (mAb) charge variants. A generic, but highly electrospray-interfering electrolyte system was used as first dimension for mAb charge variant separation and coupled to a volatile electrolyte system as second dimension via a four-port nanoliter valve. In this way, interference-free and precise mass spectrometric data of separated mAb charge variants, including deamidation products, were obtained.

Interference-free mass spectrometric detection of capillary isoelectric focused proteins, including charge variants of a model monoclonal antibody.

CIEF represents an elegant technique especially for the separation of structural similar analytes, whereas MS is a state-of-the-art instrumentation for the identification and characterization of biomolecules. The combination of both techniques can be realized by hyphenating CIEF with CZE-ESI-MS applying a mechanical valve. During the CZE step, the remaining ESI-interfering components of the CIEF electrolyte are separated from the analytes prior to MS detection. In this work, a multiple heart-cut approach is presented expanding our previous single heart-cut concept resulting in a dramatical reduction of analysis time. Moreover, different sample transfer loop volumes are systematically compared and discussed in regard to peak width and transfer efficiency. With this major enhancement, model proteins (1.63-9.75 mg/L), covering a wide pI range (5-10), and charge variants from a deglycosylated model antibody were analyzed on intact level. The promising CIEF-CZE-MS setup is expected to be applicable in different bioanalytical fields, e.g. for the fast and information rich characterization of therapeutic antibodies.

Development and validation of a capillary electrophoresis method for the characterization of sulfoethyl cellulose.

To characterize sulfoethyl cellulose el samples, a capillary electrophoresis method was developed and validated sulfoethyl cellulose el was hydrolyzed, and the resulting d-glucose derivatives were analyzed after reductive amination with 4-aminobenzoic acid using 150 mM boric acid, pH 9.5, as background electrolyte at 20°C and a voltage of 28 kV. Peak identification was derived from capillary electrophoresis with mass spectrometry using 25 mM ammonia adjusted to pH 6.2 by acetic acid as electrolyte. Besides mono-, di-, and trisulfoethyl d-glucose small amounts of disaccharides could be identified resulting from incomplete hydrolysis. The linearity of the borate buffer-based capillary electrophoresis method was evaluated using d-glucose in the concentration range of 3.9-97.5 µg/mL, while limits of detection and quantification derived from the signal-to-noise ratio of 3 and 10 were 0.4 ± 0.1 and 1.2 ± 0.3 µg/mL, respectively. Reproducibility and intermediate precision were determined using a hydrolyzed sulfoethyl cellulose el sample and ranged between 0.2 and 8.8% for migration times and between 0.3 and 10.4% for peak area. The method was applied to the analysis of the degree of substitution of synthetic sulfoethyl cellulose el samples obtained by variation of the synthetic process and compared to data obtained by elemental analysis.

CIEF-CZE-MS applying a mechanical valve.

Separation and determination of proteins by capillary isoelectric focusing (CIEF) and mass spectrometry (MS) are essential and complementary techniques in the field of bioanalysis. The hyphenation of these two techniques is challenging due to the nonvolatile substances required for the CIEF separation. An additional separation step prior to MS enables the removal of the nonvolatile substances. However, it is complicated due to the small transfer volume and the required high voltages in the CIEF process. In order to remove nonvolatile substances and transfer the analytes toward the mass spectrometer, we applied a four-port valve to couple CIEF online to capillary electrophoresis-mass spectrometry. To demonstrate the power of this concept, hemoglobin and glycated hemoglobin with an isoelectric point difference of 0.037 were separated via isoelectric focusing and characterized by MS. In general, this setup guaranties interference-free mass spectra and will provide an information-rich and sensitive top down protein characterization. Graphical abstract Interference free coupling of capillary isoelectric focusing to mass spectrometry by applying a mechanical valve. The focused proteins were tranferred from the isoelectric focusing to capillary electrophoresis by a mechanical valve. Afterwards, the transferred protein was sepearated from ionization interfering substances in the capillary electrophoresis prior to the mass spectrometry detection.

Capillary isoelectric focusing-mass spectrometry: Coupling strategies and applications.

IEF on immobilized pH gradient strips is a widespread tool for protein separation, especially as first dimension in commonly utilized 2DE. In the latter arrangement, separations are based on two orthogonal molecular characteristics according to pI in the first and molecular weight in the second dimension. However, the approach is time consuming, quantification is difficult and MS can be applied only offline. Capillary IEF and related IEF techniques in combination with MS provide similar information. The major benefits are high mass resolution and mass accuracy, reproducibility, speed, automation, and quantification by using a high-resolution mass spectrometer. However, online hyphenation of CIEF with MS is interfered by the ampholytes, acids, and bases needed for high-resolution IEF. This review will give an overview about important coupling techniques, like low ampholyte concentration, interim separation by chromatography, or the use of a dialysis interface to separate the analytes from interfering substances. It is focused on strategies which allow sensitive MS detection of CIEF-separated analytes. In addition, proteomic and biopharmaceutical applications of capillary IEF techniques combined with MS are briefly summarized.

Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection.

Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools.