SEPARATION OF HUMAN SERUM TRANSFERRIN ISOFORMS BY HIGH-PERFORMANCE PELLICULAR ANION-EXCHANGE CHROMATOGRAPHY

Glycoproteins are microheterogeneous with respect to their attached ol igosaccharides. When these oligosaccharides contain sialic acid, the o ligosaccharide microheterogeneity will impart charge heterogeneity to the glycoprotein. We found that two commercial preparations of human s erum transferrin (HST), a sialylated glycoprotein, have very different chromatographic profiles when the samples are separated by pellicular anion-exchange chromatography. Each anion-exchange profile contained multiple peaks, which suggested that both glycoproteins have charge he terogeneity. High-pH anion-exchange chromatography with pulsed amperom etric detection (HPAEC/PAD) analysis of sialic acids and oligosacchari des released from the two preparations of HST revealed that the two pr eparations differed in sialic acid and sialylated oligosaccharide cont ent. When oligosaccharides were released from the anion-exchange fract ions of the two HST preparations, the HPAEC/PAD oligosaccharide profil es showed that protein retention was directly related to sialylated ol igosaccharide content (i.e., the longer a fraction was retained, the g reater its sialylated oligosaccharide content). Therefore, the anion-e xchange profiles of the two HST preparations are related to their sial ylated oligosaccharide content. We believe that pellicular anion-excha nge chromatography can be used to quickly monitor gross changes in the sialylation of sialylated glycoproteins due to physiological state, o r in the case of recombinant glycoproteins, culture conditions and/or purification. (C) 1996 Academic Press, Inc.