MALDI linear TOF mass spectrometry of PEGylated (glyco)proteins

Abstract

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 alpha2a), 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.

Advances and challenges in analytical characterization of biotechnology products: Mass spectrometry-based approaches to study properties and behavior of protein therapeutics

“…Characterization of PEGylated biopharmaceuticals is usually focused on two questions: the extent of PEGylation ( i.e ., average number of polymer chains attached to a single protein molecule) and the location of the conjugation sites. The extent of PEGylation can be evaluated by measuring the mass of the conjugated protein by MALDI MS (Seyfried et al 2010), although pre-MS separation of conjugate forms provides more reliable results(Foser et al 2003; Lee et al 2008). …”

PEGylation of magnetic poly(glycidyl methacrylate) microparticles for microfluidic bioassays

“…For example, conventional colorimetric (TNBS, fluorometric) 21,22 and chromatographic characterization (HPLC-SEC, RP-HPLC, gel permeation chromatography) [23][24][25] in combination with light scattering, MALDI-MS 24,[26][27][28] , and capillary electrophoresis 29,30 have been applied for analysis of PEGylated proteins. Specific colorimetric detection of PEG with iodine 31, 32 using Fourier transform infrared (FTIR) or Raman spectroscopy 24 and nuclear magnetic resonance 33, 34 have also been described.…”

A New, Modular Mass Calibrant for High-Mass MALDI-MS

“…[14][15][16] High accuracy mass information is key, for example, for determining the stoi-chiometry of protein-protein, 3,4,17,18 protein-DNA, 19 or protein-RNA complexes. 20 Exact mass information is also used for the determination of the degree and the kind of post-translational modifications (PTMs), 7 PEGylations, 21 as well as for determining truncation of the primary sequence.…”