Birgit K. Seyfried1, Martina Marchetti-Deschmann1, Jürgen Siekmann2, Mary J. Bossard3, Friedrich Scheiflinger2, Peter L. Turecek2, and Günter Allmaier1
- Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria
- Baxter Innovations, Vienna, Austria
- Nektar Therapeutics, Huntsville, AL, USA
PEGylation is the most successful approach, to date, to prolong the in vivo survival of recombinant proteins. The conjugation of the polymer to glycoproteins results in challenging analysis, and furthermore, requires a wide variety of analytical tools for the determination of the extent of PEGylation. Herein, we present microchip capillary gel electrophoresis (MCGE) with a non-commercial high-molecular-weight protein assay for the analysis of the PEGylation degree with a focus on multiple PEGylation. To show the potential of the modified MCGE system, high-mass PEGylated glycoproteins (e.g. coagulation factor VIII) were analyzed. For the von Willebrand factor, the influence of glycans and the hydrodynamic radius on migration time and molecular weight determination is shown. The modified MCGE assay system is a powerful tool for the rapid assessment of the degree of PEGylation, demonstrating conjugate quality or reaction control of PEGylated proteins. This is the main advantage over time-consuming conventional SDS-PAGE. Furthermore, electrophoretic separation, staining, destaining, and fluorescence detection in one step combined with automated data analysis show that the MCGE system is a promising technique for high-throughput monitoring. The MCGE system can be used for rapid structure confirmation (“MCGE fingerprinting”) of multiply PEGylated glycoproteins beyond the 230 kDa molecular mass range.
CovalX Technology Used (Click each option to learn more)
10 mg/mL of MALDI matrix (THAP, FAc, or SA) was dissolved in acetonitrile/aqueous 1% formic acid (50:50, v/v) before 1 μL was removed and mixed with 1 μL of sample solution (in reduced form) in an Eppendorf tube. 1 μL of this final mixture was spotted onto a stainless steel MALDI target and allowed to dry at room temperature. A MALDI mass spectrometer that had been modified with a CovalX high mass detection system was used to measure the samples.