Comparison of ZIC-HILIC and graphitized carbon-based analytical approaches combined with exoglycosidase digestions for analysis of glycans from monoclonal antibodies.

Two LC approaches for analysis of therapeutic monoclonal antibodies (MAbs) are presented and compared. In the first approach, zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC) of 2-aminobenzamide-labelled glycans was coupled with fluorescence or electrospray ionisation mass spectrometric (ESI-MS) detection. The ZIC-HILIC method enabled relative quantification and identification of major glycan species. The sensitivity of fluorescence detection was higher compared to ESI-MS; however, MS detection enabled identification of co-eluted peaks. The new ZIC-HILIC approach was compared with porous graphitized carbon (PGC) separation of reduced glycans coupled with ESI-MS. Using PGC higher sensitivity was achieved compared to ZIC-HILIC due to the lower chemical background originating from the mobile phase and the derivatisation step, providing detailed information on minor glycan species. Furthermore, PGC exhibited excellent capability for separation of isobaric glycans with various degrees of mannosylation and galactosylation. The structures of glycans from MAbs used in this study were confirmed by exoglycosidase digestions. The two methods were applied to two monoclonal antibodies expressed in Chinese Hamster ovary cell lines and a monoclonal antibody expressed in a murine NS0 cell line. While the fluorescence-based approach is more suitable for routine glycan profiling due to the simplicity of data analysis, MS-based approaches were shown to provide detailed glycosylation analysis of complex glycoprotein samples.

Zwitterionic-type hydrophilic interaction nano-liquid chromatography of complex and high mannose glycans coupled with electrospray ionisation high resolution time of flight mass spectrometry.

In this study we describe a new method for rapid and sensitive analysis of reduced high mannose and complex glycans using zwitterionic-type hydrophilic interaction nano-liquid chromatography (nano ZIC-HILIC, 75 µm I.D.×150 mm) coupled with high resolution nanoelectrospray ionisation time of flight mass spectrometry (nano ESI-TOF-MS). The retention of neutral glycans increases with increasing molecular weight and is higher for high mannose glycans than for complex-type glycans. The selectivity of ZIC-HILIC for sialylated glycans differs from that for the neutral glycans and is believed to involve electrostatic repulsion; therefore, charged glycans are eluted earlier than neutral glycans with comparable molecular weight. Due to the improved sensitivity achieved by employing a ZIC-HILIC nano-column, a range of less common complex glycans has been studied and the high resolution mass spectrometry enabled confirmation of glycan composition for the proposed structures. Good sensitivity for glycans was achieved without prior fluorescent labelling, and the time of the analysis was significantly reduced compared to the separation of glycans on a conventional-size column. The proposed method offers a fast and sensitive approach for glycan profiling applied to analysis of biopharmaceuticals.

Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection.

We present a new method for the analysis of glycans enzymatically released from monoclonal antibodies (MAbs) employing a zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC) column coupled with electrospray ionization mass spectrometry (ESI-MS). Both native and reduced glycans were analyzed, and the developed procedure was compared with a standard HILIC procedure used in the pharmaceutical industry whereby fluorescent-labeled glycans are analyzed using a TSK Amide-80 column coupled with fluorescence detection. The separation of isobaric alditol oligosaccharides present in monoclonal antibodies and ribonuclease B is demonstrated, and ZIC-HILIC is shown to have good capability for structural recognition. Glycan profiles obtained with the ZIC-HILIC column and ESI-MS provided detailed information on MAb glycosylation, including identification of some less abundant glycan species, and are consistent with the profiles generated with the standard procedure. This new ZIC-HILIC method offers a simpler and faster approach for glycosylation analysis of therapeutic antibodies.

Bismuth film electrode for anodic stripping voltammetric determination of tin.

The bismuth film electrode (BiFE), in combination with anodic stripping voltammetry, offers convenient measurement of low concentrations of tin. The procedure involves simultaneous in situ formation of the bismuth film electrode on a glassy carbon substrate electrode, together with electrochemical deposition of tin, in a non-deaerated model solution containing bismuth ions, catechol as complexing agent and the metal analyte, followed by an anodic stripping scan. The BiFE is characterized by an attractive electroanalytical performance, with two distinct voltammetric stripping signals corresponding to tin, accompanied with low background contributions. Several experimental parameters were optimized, such as concentration of bismuth ions and catechol, deposition potential, deposition time and pH of the model solution. In addition, a critical comparison is given with bare glassy carbon and mercury film electrodes, revealing the superior characteristics of BiFE for measurement of tin. BiFE exhibited highly linear behavior in the examined concentration range from 1 to 100 microg L(-1) of tin (R2=0.997), an LoD of 0.26 microg L(-1) tin, and good reproducibility with a calculated R.S.D. of 7.3% for 10 microg L(-1) tin (n=10). As an example, the practical applicability of BiFE was tested with the measurement of tin in a real sample of seawater.