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Glycobiology vol 5 no 8 pp. 759-764, 1995
© 1995

research-article

Sialyl-Tn-KLH, glycoconjugate analysis and stability by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD)

P. Jorge and A. Abdul-Wajid1

Analytical Development Department, Biomira Inc. 2011-94 Street, Edmonton, Alberta, T6H 1H1, Canada

1To whom correspondence should be addressed

Received on May 9, 1995; revised on August 1, 1995; accepted on August 5, 1995

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°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 µg/ml (r2 = 0.99). The limit of quantitation (LOQ) for NANA using this isocratic method is 1.9 µg/ml ({small tilde}160 pmol/25 µl 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.

high-pH anion-exchange chromatography mono-saccharide analysis pulsed amperometric detection sialyl-Tn stability of glycoconjugate


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