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 mu g/ml (r(2) = 0.99), The limit of quantitation (LOQ) for NANA using this isocratic method is 1.9 mu g/ml (similar to 160 pmol/ 25 mu 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.