On the electromigration of charged fluorophore-labeled oligosaccharides in polyethylene oxide solutions.

The separation mechanism of charged fluorophore (aminopyrenetrisulfonate)-labeled maltooligosaccharides with α1-4 linkages was studied in polyethylene oxide (PEO) solutions (MW 300 000 Da) with special interest to possible analyte and/or network deformations as well as potential solute-matrix interactions. The electrophoretic mobilities of the 8-aminopyrene-1,3,6-trisulfonate-labeled maltooligosaccharides were found proportional with their MW(-2/3) . The Arrhenius function was used to determine the activation energy needed by the labeled sugars to migrate through the separation media.

Activation energy associated with the electromigration of oligosaccharides through viscosity modifier and polymeric additive containing background electrolytes.

The activation energy related to the electromigration of oligosaccharides can be determined from their measured electrophoretic mobilities at different temperatures. The effects of a viscosity modifier (ethylene glycol) and a polymeric additive (linear polyacrylamide) on the electrophoretic mobility of linear sugar oligomers with α1-4 linked glucose units (maltooligosaccharides) were studied in CE using the activation energy concept. The electrophoretic separations of 8-aminopyrene-1,3,6-trisulfonate-labeled maltooligosaccharides were monitored by LIF detection in the temperature range of 20-50°C, using either 0-60% ethylene glycol (viscosity modifier) or 0-3% linear polyacrylamide (polymeric additive) containing BGEs.

Effect of Separation Temperature on Structure Specific Glycan Migration in Capillary Electrophoresis.

Temperature dependent differential migration shifts were studied in capillary electrophoresis between linear (maltooligosaccharides) and branched (sialylated, neutral and core fucosylated biantennary IgG glycans) carbohydrates. Background electrolytes without as well as with low and high molecular weight additives (ethylene glycol, linear polyacrylamide and poly(ethylene oxide)) were investigated for this phenomena in the temperature range of 20-50 °C. Glucose unit (GU) value shifts were observed with increasing temperature for the all IgG glycans both in additive-free and additive-containing background electrolytes, emphasizing the importance of tight temperature control during glycosylation analysis by capillary electrophoresis.

Multicapillary SDS-gel electrophoresis for the analysis of fluorescently labeled mAb preparations: a high throughput quality control process for the production of QuantiPlasma and PlasmaScan mAb libraries.

Molecular heterogeneity of mAb preparations is the result of various co- and post-translational modifications and to contaminants related to the production process. Changes in molecular composition results in alterations of functional performance, therefore quality control and validation of therapeutic or diagnostic protein products is essential. A special case is the consistent production of mAb libraries (QuantiPlasma™ and PlasmaScan™) for proteome profiling, quality control of which represents a challenge because of high number of mAbs (>1000).

Toward the generation of an aminonaphthalene trisulfonate labeled N-glycan database for capillary gel electrophoresis analysis of carbohydrates.

There is an increasing trend to develop therapeutic glycoproteins, mostly antibodies that require high resolution bioanalytical tools to address the challenging aspects of comprehensive carbohydrate characterization. In this paper we introduce an initial version of a glucose unit database for 8-aminonaphthalene-1,3,6-trisulfonic acid-labeled glycans. At this stage we mainly focused on therapeutic IgG derived glycans of core fucosylated biantennary structures with and without sialic acid residues, as well as high mannose structures.

Rapid identification of human SNAP-25 transcript variants by a miniaturized capillary electrophoresis system.

The 25 kDa synaptosomal-associated protein (SNAP-25) is a crucial component of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and plays an important role in neurotransmission in the central nervous system. SNAP-25 has two different splice variants, SNAP-25a and SNAP-25b, differing in nine amino acids that results in a slight functional alteration of the generated soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex. Two independent techniques, a PCR-miniaturized CE method and a real-time PCR based approach were elaborated for the specific and quantitative detection of the two SNAP-25 transcription variants.

Neoglycoproteins as carbohydrate antigens: synthesis, analysis, and polyclonal antibody response.

The analysis and polyclonal antibody response for newly synthesized maltose-BSA conjugate neoglycoproteins is described. In this first proof of concept study, a simple carbohydrate antigen, maltose, was linked to BSA by reductive amination. An aglycone spacer was utilized to conserve the intact annular maltose structure and to promote the accessibility of the carbohydrate immunogen hapten during immunization.

Ultrafast haplotyping of putative microRNA-binding sites in the WFS1 gene by multiplex polymerase chain reaction and capillary gel electrophoresis.

The transmembrane protein wolframin (WSF1) plays a crucial role in cell integrity in pancreatic beta cells and maintaining ER homeostasis. Genetic variations in the WFS1 gene have been described to be associated with Wolfram syndrome or type 2 diabetes mellitus. In this paper we report on an efficient double-tube allele-specific amplification method in conjunction with ultrafast capillary gel electrophoresis for direct haplotyping analysis of the SNPs in two important miRNA-binding sites (rs1046322 and rs9457) in the WFS1 gene.

Light-emitting diode induced fluorescence (LED-IF) detection design for a pen-shaped cartridge based single capillary electrophoresis system.

CGE is a well-established separation technique for the analysis of biologically important molecules such as nucleic acids. The inherent high resolving power, rapid analysis times, excellent detection sensitivity, and quantification capabilities makes this method favorable compared to conventional manual polyacrylamide and agarose slab gel electrophoresis techniques. In this paper we introduce a novel single-channel capillary gel electrophoresis system with LED-induced fluorescence detection also utilizing a compact pen-shaped capillary cartridge design for automatic analysis of samples from a 96-well plate.

High-throughput analysis of therapeutic and diagnostic monoclonal antibodies by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling.

Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is a well-established and widely used protein analysis technique in the biotechnology industry, and increasingly becoming the method of choice that meets the requirements of the standards of International Conference of Harmonization (ICH). Automated single channel capillary electrophoresis systems are usually equipped with UV absorbance and/or laser-induced fluorescent (LIF) detection options offering general applicability and high detection sensitivity, respectively; however, with limited throughput. This shortcoming is addressed by the use of multicapillary gel electrophoresis (mCGE) systems with LED-induced fluorescent detection (LED-IF), also featuring automation and excellent detection sensitivity, thus widely applicable to rapid and large-scale analysis of biotherapeutics, especially monoclonal antibodies (mAb).