Analysis of charge heterogeneities in mAbs using imaged CE.

An analytical method using the imaged CE (iCE) technology has been developed and validated for measuring contents of charge variants for mAb molecules. The method could generate similar information that require both conventional IEF electrophoresis and ion exchange liquid chromatography to generate as an iCE analysis would produce both the pI of the molecules and quantitative contents of charge variants. Thus, it offers unique advantages over the IEF and ion exchange methods in terms of identification, separation and quantitation of charge variants. The data presented in this study demonstrated that the iCE method is suitable not only for research purposes but also for quality control purpose of mAb clinical or commercial manufacture as the technique can be validated and possesses sufficient robustness. The developed generic iCE method has been tested for a wide range of therapeutic mAbs and proved its suitability for multiple mAb molecules. The tested mAbs are a group of molecules with a wide range of charge compositions (acidic species ranging from 10 to 70%) and a diverse range of pI of 6.9-9.6. Developing platform analytical technologies is an efficient way to meet the demand of rapid growth of therapeutic mAb candidates under clinical and preclinical development. The iCE technology is a good candidate to become a platform method as it could cover a broad range of pH gradient. The experiences learned during the developmental process would provide important and valuable information to the biotech industry for the evaluation of charge heterogeneity of mAbs in terms of release testing, characterization, stability study, process development support and comparability study.

Coupling capillary electrochromatography with electrospray Fourier transform mass spectrometry for characterizing complex oligosaccharide pools.

To deal with the complexity of the glycan mixtures released from glycoproteins, an efficient form of micro-column separations, capillary electrochromatography, has been combined with high-performance mass spectrometry (Fourier transform ion cyclotron resonance). Contour plots have been generated from the mixtures of O-linked oligosaccharides originated from bovine mucin and bile salt-stimulated lipase, a large glycoprotein enzyme.

Structural characterization of neutral oligosaccharide mixtures through a combination of capillary electrochromatography and ion trap tandem mass spectrometry.

A CEC/ESI-MS/MS combined system has been developed for the separation and on-line structural analysis of neutral oligosaccharides. Various types of isomeric oligosaccharides were first successfully separated by CEC using polar monolithic columns, while the on-line tandem mass spectrometry has been explored to differentiate and elucidate the structures of isomeric oligosaccharides. The experimentally obtained tandem spectra usually provide sequence, branching, and linkage information. Oligosaccharide isomers with a different monomeric composition and branching showed different patterns of glycosidic linkage cleavage (B- and Y-ion series), allowing us to deduce their sequence and branching points. Isomers with different linkages were distinguished by identifying cross-ring fragment ions (A-ion series). While (1-->4) linkages yielded dominant (0,2)A ions, (1-->6) linkages showed an extensive and complete cross-ring cleavage series: (0,2)A, (0,3)A, and (0,4)A ions. Although the anomeric configurations and monosaccharide identification are rarely obtained from tandem MS, the relevant mixture components can be completely resolved with high-efficiency CEC columns featuring a polar functionality.

Separation of neutral saccharide mixtures with capillary electrochromatography using hydrophilic monolithic columns.

While developing a combination of capillary electrochromatography (CEC) with tandem mass spectrometry (MS) for the benefit of characterizing complex oligosaccharide mixtures, we needed highly efficient CEC columns operating in an "MS-friendly" mode. We demonstrate here novel types of polar, monolithic CEC columns that separate effectively complex mixtures of saccharides with the use of mobile phases containing acetonitrile/dilute ammonium formate buffers. Using the positive-ion mode of detection for neutral saccharides, the detection conditions were optimized down to the low-femtomole sensitivities with the use of an ion trap mass spectrometer. This column technology provides a nearly universal system that can separate a wide range of carbohydrates: mono- and oligosaccharides with the intact reducing end, as well as saccharide alditols. Even the anomers formed due to mutarotation could be resolved with a high content of organic phase.