Separating glycoproteins using WAX chromatography

November 7, 2011

The Column

The Column, The Column-11-07-2011, Volume 7, Issue 20

The separation of intact glycoproteins presents a great challenge because one glycoprotein can exhibit many isoforms with close physiochemical properties. As a result, it is difficult to study them in isolation. A team of chemists from Nanjing University, China, have tested a polymeric weak anion exchange (WAX) monolithic capillary to assess the high resolution separation of glycoprotein isoforms, to see whether this provides the desired results.

The separation of intact glycoproteins presents a great challenge because one glycoprotein can exhibit many isoforms with close physiochemical properties. As a result, it is difficult to study them in isolation. A team of chemists from Nanjing University, China, have tested a polymeric weak anion exchange (WAX) monolithic capillary to assess the high resolution separation of glycoprotein isoforms, to see whether this provides the desired results.1

First they prepared a base monolith through ring-opening polymerization between tris(2,3-epoxypropyl)isocyanurate and tri(2-aminoethyl). This was then modified through a reaction with an aqueous ammonia solution to convert the unreacted epoxide moieties into primary amino groups. The prepared monolithic capillary was characterised by morphology, pore size, hydrophilicity and reproducibility.

Various versions of the monolith were produced by changing the ratio of the two molecules, the temperature of the reaction and the concentration of the pore-generating molecule. The performance of the monolith capillary was then evaluated using several typical glycoproteins as mode analytes. Under the optimized separation conditions, the tested glycoproteins were all resolved into distinct glycoforms. The monolith’s separation ability was compared with capillary zone electrophoresis (CZE) and revealed that the WAX column separated more glycoforms, both consistently and at a faster speed, although the resolution of some glycoprotein isoforms decreased.

Reference

1 Zhen Liu et al., J. Chromatogr. A, September 2011, doi: 10.1016/j. chroma 2011.08.079

This story originally appeared in The Column. Click here to view that issue.