The use of sequential high-performance liquid chromatography and capillary zone electrophoresis to separate the glycosylated peptides from recombinant tissue plasminogen activator to a detailed level of microheterogeneity.

The advantage of using the two-dimensional separation method was demonstrated by the separation of the complex tryptic peptides of recombinant human tissue plasminogen activator (rt-PA) glycoprotein. This method hybridizes two analytical methods where fractions containing glycopeptides are collected from reversed-phase high-performance liquid chromatography (RP-HPLC) and separated by capillary zone electrophoresis (CZE). CZE readily resolved carbohydrate structural variants, based on sialic acid content and branching, on the same peptide. Nonglycosylated peptides incidentally collected in the same RP-HPLC fraction were well resolved from the glycopeptides. This combination of RP-HPLC and CZE was able to uniquely resolve all of the peptides and glycopeptide variants in rt-PA. Confirmation of the specific peaks in the CZE was made by matrix-assisted laser-induced ionization time-of-flight mass spectrometry. The advantages and disadvantages of the existing methods for carbohydrate characterization in the biotech industry were compared. The advantage of this approach is to provide a simple extension of the existing tryptic digest protocols to include carbohydrate analysis to a detailed level of microheterogeneity.