title: Reaction of Oxidized Polysialic Acid and a Diaminooxy Linker: Characterization and Process Optimization Using Nuclear Magnetic Resonance Spectroscopy.
abstract: Native polysialic acid (natPSA) is a high-molecular-weight glycan composed of repeat units of α-(2 → 8) linked N-acetylneuraminic acid (Neu5Ac). Mild periodate oxidation of PSA selectively targets the end sialic acid ring containing three adjacent alcohols generating a putative aldehyde, which can be used, after attachment of a linker molecule, for terminal attachment of PSA to protein. Previously, we showed that the oxidized PSA (oxoPSA) contained a hemiacetal at the oxidation site and can react with a linker containing an aminooxy group in a conjugation reaction to form a stable oxime linkage. Thus, reagents containing an aminooxy group may be prepared for conjugation of PSA to the carbohydrate moiety of therapeutic proteins, thereby increasing their half-life. These aminooxy-PSA reagents can selectively react with aldehyde groups generated by mild NaIO4 oxidation of glycans on the surface of the target protein. To comprehend the conjugation, unoxidized tetrasialic acid and Neu5Ac were reacted in model reactions with a diaminooxy linker to define the nuclear magnetic resonance (NMR) chemical shifts. Based on these data, we were able to show that, in the case of PSA, the reaction with the linker occurs not only at the expected oxidized end to form an aldoxime but also at the end distal to the oxidation to form a ketoxime. We determined that, in aged solutions, both oxoPSA and PSA aldoxime were hydrolyzed. PSA aldoxime was also shown to disproportionate to form a dimer (PSA-linker-PSA), which then could react further with the released linker at one of its PSA termini. Furthermore, NMR was used to monitor the effects of deliberate process changes so that conditions could be optimized for attachment of linker at the desired end of the PSA chain, which led to a well-defined product.
authors: Baxter International Inc. , 25212 West Illinois Route 120, Round Lake, Illinois 60073, United States.
authors: G Joseph
authors: GJ
authors: Ray
authors: Baxalta Innovations GmbH, now part of Shire , Industriestraße 67, Vienna, A-1221, Austria.
authors: Jürgen
authors: J
authors: Siekmann
authors: Baxalta Innovations GmbH, now part of Shire , Industriestraße 67, Vienna, A-1221, Austria.
authors: Richard
authors: R
authors: Scheinecker
authors: Soochow University , College of Pharmaceutical Sciences, 199 Ren'ai Road, Suzhou, Jiangsu 215123, China.
authors: Zhenqing
authors: Z
authors: Zhang
authors: Baxter International Inc. , 25212 West Illinois Route 120, Round Lake, Illinois 60073, United States.
authors: Mikhail V
authors: MV
authors: Gerasimov
authors: Baxter International Inc. , 25212 West Illinois Route 120, Round Lake, Illinois 60073, United States.
authors: Christina M
authors: CM
authors: Szabo
authors: University of Natural Resources and Life Sciences , Department of Chemistry, Muthgasse 18, Vienna, A-1190, Austria.
authors: Paul
authors: P
authors: Kosma
mesh_terms: D000447:Aldehydes / Q000737:chemistry; D007659:Ketones / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D010091:Oximes / Q000737:chemistry; D012794:Sialic Acids / Q000737:chemistry*
chemical_list: D000447:Aldehydes; D007659:Ketones; D010091:Oximes; D012794:Sialic Acids; C021319:polysialic acid