Aldehyde PEGylation Kinetics: A Standard Protein versus a Pharmaceutically Relevant Single Chain Variable Fragment

Abstract

The mPEG-aldehyde PEGylation with two different PEG sizes and two proteins was experimentally determined with respect to yield, conversion, and selectivity. The kinetic behavior of these PEGylation reactions was simulated using a numerically solved set of differential equations. We show that the assumption of an inactivation of mPEG-aldehyde is crucial for the simulation of the overall PEGylation and that the inactivation is pH-dependent. We further demonstrate that ideal PEGylation parameters such as pH, temperature, reaction time, and protein concentration need to be chosen carefully depending on the protein and PEG size. In terms of selectivity and yield, we show that the reaction should be stopped before the highest mono-PEG concentration is reached. Moreover, room temperature and a slightly acidic pH of approximately 6 are good starting points. In conclusion, selectivity can be optimized choosing a shorter reaction time and a reduced reaction temperature.

Kinetic modeling of protein PEGylation

“…The high reactivity of the functionalized PEG goes in fact along with the hydrolysis of its active moiety which cannot be neglected. Moosmann et al (Moosmann et al, 2011) showed that accurate simulations are not possible without considering this deactivation reaction. Esters hydrolysis mechanisms have been thoroughly investigated over the years and appear to be pH-dependent, affected by both basic and acidic conditions (Day and Ingold, 1941).…”

“…Their kinetic model is easy to implement and represents an effective tool to simulate the formation of PEGylated proteins at various degrees of PEGylation (also named PEGamers). This model has already been used with some success 3 in the literature (Molek, 2008;Moosmann et al, 2011;Wang et al, 2014). The main limitation of the approach proposed by Laura et al is that the employed kinetic model does not allow the distinction between the various positional isomers that compose PEGamers of a given degree of PEGylation (doP).…”

Process development of periplasmatically produced single chain fragment variable against epidermal growth factor receptor in Escherichia coli

“…The in vivo half-life of this pharmaceutically relevant target protein, used in this study, was increased by the covalent coupling of polyethylene glycol polymer chains (PEGylation) previously described by Moosmann et al (2011Moosmann et al ( , 2012. Like the parental chimeric (mouse/human) monoclonal IgG1 antibody Cetuximab, which is an FDA approved pharmaceutical drug, the humanized scFv is a competitive antagonist for EGF, which could be used in the treatment of metastatic colorectal cancer (Vincenzi et al, 2008;Boder and Jiang, 2011).…”

Monitoring lactoferrin iron levels by fluorescence resonance energy transfer: a combined chemical and computational study

“…The lysine residues on the external surface of lactoferrin can be used to covalently couple molecules [19][20][21][22] [24][25][26].…”