Investigation of a side reaction occurring during N-linked glycan labeling by cationic tags.

Capillary electrophoresis-mass spectrometry was applied for the analysis of oligosaccharides and N-linked glycans with an attached charge label facilitating electrophoretic migration and electrospray ionization efficiency. Several different labeling strategies have been tested with different tags and tagging reactions including reductive amination and hydrazone formation. However, a formation of multiple labeled N-linked glycans was observed by CE-MS in a positive ion mode when positively charged labels such as aliphatic amines containing a quaternary ammonium group were attached to N-linked glycans by reductive amination. A reaction mechanism explaining a side reaction occurring during the labeling and the multiple product formation was proposed and confirmed by using isotopically labeled N-acetylglucosamine. Finally, it was confirmed that derivatization of sugars via a hydrazone formation can be a simpler method with a high reaction yield suitable for high sensitive CE-ESI/MS analyses of N-linked glycans.

Multi-charged labeling of oligosaccharides and N-linked glycans by hexahistidine-based tags for capillary electrophoresis-mass spectrometry analysis.

The labeling by amino acids and peptides was investigated for sensitive and fast analyses of oligosaccharides and N-linked glycans by capillary electrophoresis-mass spectrometry (CE-MS). Peptide tags with a various number of histidine residues were tested for maltooligosaccharide labeling in order to investigate the effect of the size of labels and a number of charges on CE-MS analysis. Nevertheless, the reductive amination labeling of N-linked glycans by a hexahistidine tag resulted in a multiple products formation, therefore a peptide tag was modified by hydrazine functionality in order to perform labeling by hydrazone formation chemistry. This labeling approach significantly improved sensitivity with LOD of labeled maltopentaose determined to be 40 nmol/L and also significantly reduced separation time of neutral maltooligosaccharides and N-linked glycans released from bovine ribonuclease B. Furthermore, the labeling by this multi-cationic peptide hydrazine tag also allowed performing analysis of acidic glycans by CE-MS in a positive ion mode as demonstrated by separation of sialylated N-linked glycans released from bovine fetuin.

Sample preparation for N-glycosylation analysis of therapeutic monoclonal antibodies by electrophoresis.

There are a considerable number of biopharmaceuticals that have been approved for clinical use in the past decade. Over half of these new generation drugs are glycoproteins, such as monoclonal antibodies or other recombinant glycoproteins, which are mostly produced in mammalian cell lines. The linked carbohydrate moieties affect not only their physicochemical properties and thermal stability but also crucial features like receptor-binding activity, circulating half-life, as well as immunogenicity. The structural diversity of these attached glycans can be manifested in altered monosaccharide composition and linkages/positions among the monosaccharide building blocks. In addition, as more and more biosimilar products hit the market, understanding the effects of their glycosylation modification has become a recent target in efficacy and safety issues. To ensure consistent quality of these products, glycosylation profiles have to be monitored and controlled in all steps of the manufacturing process, i.e., from clone selection to lot release. In this paper, we describe some of the recently introduced and commonly used sample preparation techniques for capillary electrophoresis (CE)-based profiling and structural elucidation of N-glycans. The presented protocols include protein A affinity partitioning of monoclonal antibodies (mAbs), enzymatic release of the N-linked glycans, labeling of the liberated carbohydrates, reaction mixture purification techniques to remove the excess labeling reagent, and high-resolution and rapid capillary electrophoresis-laser-induced fluorescence (CE-LIF)-based profiling of the labeled and purified N-glycans.

Cationic labeling of oligosaccharides for electrophoretic preconcentration and separation with contactless conductivity detection.

Cationic derivatization of oligosaccharides by quaternary ammonium label was investigated for capillary electrophoretic separation with transient isotachophoretic preconcentration (t-ITP) as detected by capacitively coupled contactless conductivity detection (C4D). Dextran ladder, prepared by partial hydrolysis of dextran, isomaltotriose, maltopentaose and maltoheptaose were derivatized by reductive amination with (2-aminoethyl)trimethylammonium chloride. This label provides permanent positive charge potentially also useful for mass spectrometry ionization. Capillary electrophoresis (CE) separations of the oligosaccharides were tested in zone electrophoretic (CZE) and combined t-ITP/CZE modes. The transient isotachophoretic preconcentration was performed by injecting the sample solution complemented by ammonium acetate into the acetic acid background electrolyte. In this case the ammonium ions served as leading electrolyte followed by the sample zone and the background electrolyte of acetic acid acted as terminating electrolyte. The oligosaccharides were focused into narrow zones by ITP principles and during the course of migration through the separation capillary relaxed into zone electrophoretic separation mode. The separated ones were detected by C4D detector with detection limits in the nanomolar concentration range.