Sialyl-Tn-KLH, glycoconjugate analysis and stability by high-pH anion-exchange chromatography with pulsed...

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.