Notes

Oligosaccharides Chromatography Detection
Understanding and characterizing protein therapeutic glycosylation is important with growing evidence that glycosylation impacts biological efficacy, pharmacokinetics and cellular toxicity. Protein expression systems and reactor conditions can impact glycosylation, leading to potentially undesirable glycosylation. For lacto n neotetraose , high-mannose species may be present, which are atypical of human antibody glycosylation. Their presence in the Fc domain has been linked to increased serum clearance of immunoglobulin G (IgG) antibodies. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an effective tool for determining glycans present in glycoprotein therapeutics. We report an improved HPAE-PAD method for IgG oligosaccharide separation.

The neutral glycans are well resolved, including separation of high-mannose species from typical human IgG glycans. Oligosaccharide identification was performed by comparison to known standards in conjunction with selective exoglycosidase digestion of both standards and released glycans. Retention times (RTs) of known glycans were compared with the retention times of maltose, maltotriose and maltotetraose standards to define a retention index value for each glycan. These retention indices were used to aid identification of glycans from an example monoclonal antibody sample of unknown glycosylation. Method ruggedness was evaluated across duplicate systems, analysts and triplicate column lots. Comparing two systems with different analysts and columns, retention time precision relative standard deviations from7 to6%. The separation is orthogonal to capillary electrophoresis-based separation of labeled IgG oligosaccharides.

Translocation of Oligonucleotide-Oligosaccharide Complexes into Cells of the Sciences, Novosibirsk, 600, Russia.Irradiation of a mixture of oligonucleotides with dextran resulted in the formation of a complex that is detected by a decelerated migration of an irradiated sample in electrophoretic gel compared to a non-irradiated one. When injected into the brain of neonatal rats, the formed complex penetrated into the cells 3 times easier compared to the original oligonucleotide, thus indicating that the use of radiation crosslinking of oligonucleotides with oligosaccharides is promising to enhance the efficiency of delivery of gene-targeted Sulfated sialyl-oligosaccharides derived from tracheobronchial mucous glycoproteins of a patient suffering from cystic fibrosis.Thirteen novel oligosaccharides, each possessing both a sulfate ester and a sialic acid residue, were isolated from tracheobronchial mucous glycoproteins from a patient with cystic fibrosis via cleavage by alkaline borohydride treatment, and by employing immobilized Limulus polyphemus lectin affinity chromatography, SynChroprep AX0 anion-exchange chromatography, Bio-Gel P-2 size-exclusion chromatography, and Hypersil 1A APS-2 high-performance liquid chromatography (HPLC). Proposed structures for the resulting purified sulfated sialyl-oligosaccharides were based on carbohydratepermethylation analyses, periodate oxidation, complete sequential exoglycosidase digestion, analysis of desulfated products and, analysis by positive-ion fast-atom-bombardment mass spectrometry (FABMS). Lactose-N-neotetraose on these sialyl-oligosaccharides resided on C-6 of a terminal or an internal D-galactose or 2-acetamido-2-deoxy-D-glucose residue or C-4 of a terminal D-galactose residue. The sialic acid residues were found to be either bound (2--6)-alpha to 2-acetamido-2-deoxy-D-galactitol or terminus.

For this group of oligosaccharides, ranging in size from tri- to hepta-saccharides, it was also observed that a sialic acid residue and a sulfate ester did not residue on the same oligosaccharide branch when more than one branch existed. On linear unbranched sulfated sialyl-oligosaccharides, the sialic acid residue was bound to a D-galactose residue occupying a nonreducing terminus with the sulfate ester residing on an internal D-galactose or a 2-acetamido-2-deoxy-D-glucose residue. These results demonstrate that it is possible for sialic acid and a sulfate ester to exist on the same oligosaccharide and that this oligosaccharide can be as small as a Pyruvylated glycolipids from Mycobacterium smegmatis. Structures of two A crude glycolipid fraction was isolated from Mycobacterium smegmatis ATCC 356 by ethanolic extraction and silica gel chromatography. After deacylation of the glycolipid fraction, a dipyruvylated pentasaccharide (acidic oligosaccharide A) and a monopyruvylated tetrasaccharide (acidic oligosaccharide B1) were purified by ion exchange and gel filtration chromatography.
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