Oligosaccharide Release and MALDI-TOF MS Analysis of N-Linked Carbohydrate Structures from Glycoproteins

Production, characterization and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans

“…Selected samples were confirmed using a well-established matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric glycan characterization assay. 35,36 In the rpHPLC ESI-MS method, the distribution of glycans attached to the antibody was deduced from the overall mass of the heavy chain with its associated N-glycan. This method does not require release of glycan using N-glycosidase F (PNGaseF), 35,36 thereby allowing rapid characterization of the antibody glycoform.…”

“…35,36 In the rpHPLC ESI-MS method, the distribution of glycans attached to the antibody was deduced from the overall mass of the heavy chain with its associated N-glycan. This method does not require release of glycan using N-glycosidase F (PNGaseF), 35,36 thereby allowing rapid characterization of the antibody glycoform. To prepare the samples for analysis, 0.1 to 0.5 mg of antibody was mixed with a 1:1 (w/w) ratio of reducing agent tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and solvent (10% acetonitrile/0.1% formic acid/HPLC-grade water) to reach a final antibody concentration of 0.5-1 mg/mL.…”

Evolutionarily Conserved Paired Immunoglobulin-like Receptor α (PILRα) Domain Mediates Its Interaction with Diverse Sialylated Ligands

“…Samples were acidified with glacial acetic acid and desalted using a Dowex 50X8 cation-exchange resin spin column (25). O-Glycans were collected in the flow-through.…”

“…Permethylated glycans were cleaned via solid phase extraction on a vacuum-driven Hypersep C18 10-mg cartridge (Thermo Scientific), followed by repeated washes with water and 5% aqueous solution of acetonitrile, and elution with a 75% aqueous solution of acetonitrile. For MALDI-TOF MS analysis, 1 l of permethylated glycans was spotted on the stainless steel target, followed by 1 l of "super 2,5-dihydroxybenzoic acid" matrix, and samples were briefly dried using vacuum (25). Spectra were acquired in a positive mode on a Bruker UltraFlex MALDI-TOF/TOF instrument and calibrated with permethylated glucose homopolymer mixture (ProZyme).…”

Rapid characterization of N‐linked glycans from secreted and gel‐purified monoclonal antibodies using MALDI‐ToF mass spectrometry

“…This would allow increased profiling of antibodies from multiple cell lines during culture and facilitate better optimization of production processes to make quality integral to the system, a facet which is essential for process analytical technology (PAT). Methods have been published for the analysis of glycans in multi-well formats using PVDF coated 96-well plates for the non-specific capture and concentration of protein (Keck et al, 2005;Papac et al, 1998). The workflows described below enable the specific purification and concentration of the antibody prior to glycan analysis.…”