Sialyl-Tn-KLH, glycoconjugate analysis and stability by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD).
The quantitation of sialyl-Tn (STn) conjugated to keyhole limpet haemocyanin (KLH) can be determined by quantitating the amount of N-acetylneuraminic acid (NANA) released by acid or enzymatic digestion. An optimal 0.1 N H2SO4 acid hydrolysis at 80 degrees C results in quantitative release of NANA with minimal loss. A rapid isocratic method for the quantitation and separation of NANA is described using high-pH anion-exchange chromatography and pulsed amperometric detection (PAD). Multiple injection of NANA standard and/or samples containing protein led to a decrease in the PAD response which was corrected by addition of internal standard, alpha-2-keto-3-deoxyoctonate (KDO). The ratio of NANA/KDO peak area or peak height gives a linear response with increasing amount of NANA in the range 2.5-20 micro g/ml (r2 = 0.99). The limit of quantitation (LOQ) for NANA using this isocratic method is 1.9 micro g/ml (approximately 160 pmol/25 micro l injection). Based on the multiple determination the glycoconjugate, STn-KLH, showed a NANA content of 2.9% (w/w). Acid hydrolysis and the sialidase treatment of STn-KLH both yielded a similar NANA content. The carrier protein, KLH, showed the absence of NANA. The stability of glycoconjugate STn-KLH was monitored by a gradient method which separated possible degradation products STn-crotyl, NANA and GalNAc. Subjecting the glycoconjugate STn-KLH to various stress conditions of temperature, pH and oxidation does not result in any release of sialic acid, GalNac and STn-crotyl group.
Jorge
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P
Analytical Development Department, Biomira Inc., Edmonton, Alberta, Canada.
Abdul-Wajid
A
A
D015295:Antigens, Tumor-Associated, Carbohydrate / Q000737:chemistry* / Q000378:metabolism; D019496:Cancer Vaccines / Q000737:chemistry* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D006433:Hemocyanins / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D019158:N-Acetylneuraminic Acid; D009439:Neuraminidase / Q000378:metabolism; D012794:Sialic Acids / Q000032:analysis*; D017186:Titrimetry
D015295:Antigens, Tumor-Associated, Carbohydrate; D019496:Cancer Vaccines; D012794:Sialic Acids; C099077:sialyl-Tn antigen-keyhole-limpet hemocyanin conjugate; D006433:Hemocyanins; D009439:Neuraminidase; C032808:keyhole-limpet hemocyanin; D019158:N-Acetylneuraminic Acid