Gal Alpha Gal Analysis
This assay measures galactose linked α1-3 to another galactose molecule on recombinant glyco-proteins and other glycans. Galactose is released; enzymatically using an enzyme specific for galactose in α1-3 linkage and then free galactose is identified by HPLC (high performance liquid chromatography) and measured by comparing the response in the sample to a standard curve of galactose.
Identification of the oligosaccharide structures on glycoprotein samples are accomplished by isolating the different oligosaccharides often by collecting peaks from an oligosaccharide profile and characterizing them using methods such as HPLC, MALDI-TOF etc.
Glycosidase sequencing selectively releases monosaccharides from the oligosaccharide. This can also be used to determine their linkages.
Mannose 6-Phosphate Analysis
This assay measures mannose 6-phosphate (Man 6-P/M6P) on recombinant glycoproteins. Mannose 6-phosphate is released from glycoproteins using acid hydrolysis. Man 6-P is then identified by HPLC (high performance liquid chromatography) and measured by comparing the response in the sample to a standard curve of Man 6-P
Neutral Monosaccharide Composition
This analysis measures the most common neutral monosaccharides (fucose, N-acetylglucosamine, N-acetylgalactosamine, mannose and galactose) in samples although we are also able to quantify additional monosaccharides such as glucose and xylose. We have separate SOPs that have been optimized for MAbs or general glycoproteins.
Samples are hydrolyzed in acid and then analyzed by HPAEC (high pH anion-exchange chromatography) using electrochemical detection. The monosaccharides are quantified relative to standard curves generated for each monosaccharide. The data are usually expressed as moles monosaccharide/mole protein.
N and O-linked Oligosaccharide Profiling
By using a chemical method that releases both N and O-linked oligosaccharides we are able to generate a profile that includes both forms of oligosaccharides. The released oligosaccharides are labeled at their reducing end (the end that was attached to the protein) with the fluorophore 2-aminobenzoic acid (AA). The labeled oligosaccharides are then separated by HPLC or analyzed by MALDI-TOF Mass Spectrometry.
HPAEC (High performance Liquid Anion-Exchange Chromatography), with Pulsed Amperometric Detection (PAD) and MALDI-TOF-MS analysis were employed for the identification and analysis of complex mano and oligosaccharides.
Sialic Acid Analysis
This assay quantifies the two most common sialic acids: N-acetylneuraminic acid (NeuAc/NANA) and N glycolylneuraminic acid (NeuGc/NGNA) on glycoproteins. Sialic acids need to be analyzed separately from neutral monosaccharides because they are more acid labile than neutral monosaccharides and are destroyed by the acid hydrolysis conditions required to release neutral monosaccharides from the glycoprotein.
Samples are hydrolyzed in mild acid and then analyzed by HPAEC (high pH anion-exchange chromatography) using electrochemical detection. The sialic acids are quantified relative to standard curves of each sialic acid. The data are usually expressed as moles sialic acid/mole protein.