Birgit K. Seyfried1, Jürgen Siekmann2, Omar Belgacem3, Ryan J. Wenzel4, Peter L. Turecek2, and Günter Allmaiera1
- Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria
- Baxter Innovations, Vienna, Austria
- Shimadzu Biotech Kratos Analytical, Manchester, UK
- CovalX, Zurich, Switzerland
PEGylation of proteins is a fast growing field in biotechnology and pharmaceutical sciences owing to its ability to prolong the serum half-life time of recombinant proteins. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) has been shown to be a powerful tool in the analysis of several PEGylated small proteins. Here we present data obtained with a standard secondary electron multiplier (SEM) and a high mass (HM) detector combined with a MALDI linear TOF MS system for the detection of PEGylated (glyco)proteins in the range of 60-600 kDa. Examples of MALDI TOF MS of small (interferon α2a), middle (human serum albumin (HSA)) and high molecular mass proteins (coagulation factor VIII and von Willebrand factor (vWF), both heavily glycosylated proteins) are presented. The particular challenge for the analysis was the heterogeneity of the (glyco)proteins in the high molecular weight range in combination with additional PEGylation, which even introduced more heterogeneity and was more challenging for interpretation. Nevertheless, the performance of MALDI linear TOF MS with both detector systems in terms molecular weight and heterogeneity determination depending on the m/z range was superior to the other methods. Although the SEM was able to obtain information about protein PEGylation in the mass range up to 100 kDa (e.g. PEGylated HSA), the HM system was crucial for detection of HM ions (e.g. PEGylated recombinant vWF), which was impossible with the standard SEM.
CovalX Technology Used (Click each option to learn more)
A matrix was created using 10 mg/ml of sinapic acid dissolved in acetonitrile:1% aqueous formic acid (1:1, v/v). 0.5 μL of matrix was placed on a MALDI target and then the sample solution (PEGrIFN, HSA or PEGHSA) was injected into the droplet before being allowed to dry at room temperature. 1 μL of matrix and 1 μL of sample solution (rFVIII and rvWF dimer in PEGylated and non-conjugated form) were mixed in an Eppendorf tube and then 1 μL of the final mixture applied to a stainless steel MALDI target and allowed to dry at room temperature. The targets were analyzed by a mass spectrometer that had been modified with a CovalX HM1 detection system. Through the use of this system, researchers were able to determine the molecular weight of most of the samples.