PEGylated recombinant von Willebrand factor analyzed by means of MALDI-TOF-MS, CGE-on-a-chip and nES-GEMMA

Recombinant VWF (rVWF) is a candidate for therapy of von Willebrand disease and the largest known multimeric glycoprotein. In this study rVWF was covalently linked with a 20 kDa branched polyethylene glycol (PEG) to obtain PEGylated rVWF (PEGrVWF). This conjugation results in a further increase of heterogeneity besides glycoheterogeneity and a challenge in analyzing of such a bioconjugate, particular when investigated on the intact molecule level. Four different techniques including SDS-PAGE, MALDI-TOF-MS, capillary-gel-electrophoresis-on-a-chip (CGE-on-a-chip) and nano electrospray gas-phase electrophoretic mobility molecular analysis (nES-GEMMA) were applied to determine the molecular weight (MW) and the PEGylation degree of the monomeric rVWF. The degree and distribution of PEGylation of rVWF obtained by CGE-on-a-chip were in good agreement with results obtained by MALDI-TOF-MS with a special high mass detector. An average PEGylation degree of 3.1 PEG chains coupled to the monomeric glycoforms was found. MW determination by MALDI-TOF-MS (317.4 ± 1.0 kDa; 3 PEG chains attached) showed in comparison to CGE-on-a-chip (413.4 ± 2.1 kDa) the highest precision. Furthermore the orthogonal method, nES-GEMMA provided first information on the globular size (11.3 ± 0.1 nm) and based on that the MW (251 ± 7.2 kDa for the average PEGylation) of the PEGrVWF.

Figure optionsDownload high-quality image (123 K)Download as PowerPoint slideResearch highlights▶ Von Willebrand factor (VWF) – largest known human multimeric glycoprotein (up to 20 MegaDa). ▶ Candidate for therapy of Von Willebrand disease. ▶ Extrem heterogeneity of PEGylated glycoprotein – recombinant VWF. ▶ Molecular mass and degree of PEGylation of multiple PEGylated VWF by MALDI-MS and CGE-on-a-chip. ▶ Size determination PEGylated recombinant VWF by nES-GEMMA.