TY - JOUR
T1 - Large-scale biophysical evaluation of protein PEGylation effects
T2 - in vitro properties of 61 protein entities
AU - Vernet, Erik
AU - Popa, Gina
AU - Pozdnyakova, Irina
AU - Rasmussen, Jakob Ewald
AU - Grohganz, Holger
AU - Giehm, Lise
AU - Jensen, Malene Hillerup
AU - Wang, Huabing
AU - Plesner, Bitten
AU - Nielsen, Hanne Mørck
AU - Jensen, Knud Jørgen
AU - Berthelsen, Jens
AU - Sundström, Michael
AU - van de Weert, Marco
PY - 2016
Y1 - 2016
N2 - PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but surprisingly limited public data is available on the biophysical effects of protein PEGylation. Here we report the first large-scale study, with site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total using a common set of analytical methods. Predictions of molecular size were typically accurate in comparison with actual size determined by size-exclusion chromatography (SEC) or dynamic light scattering (DLS). In contrast, there was no universal trend regarding the effect of PEGylation on the thermal stability of a protein based on data generated by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF). In addition, DSF was validated as a fast and inexpensive screening method for thermal unfolding studies of PEGylated proteins. Multivariate data analysis revealed clear trends in biophysical properties upon PEGylation for a subset of proteins, although no universal trends were found. Taken together, these findings are important in the consideration of biophysical methods and evaluation of second-generation biopharmaceutical drug candidates.
AB - PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but surprisingly limited public data is available on the biophysical effects of protein PEGylation. Here we report the first large-scale study, with site-specific mono-PEGylation of 15 different proteins and characterization of 61 entities in total using a common set of analytical methods. Predictions of molecular size were typically accurate in comparison with actual size determined by size-exclusion chromatography (SEC) or dynamic light scattering (DLS). In contrast, there was no universal trend regarding the effect of PEGylation on the thermal stability of a protein based on data generated by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF). In addition, DSF was validated as a fast and inexpensive screening method for thermal unfolding studies of PEGylated proteins. Multivariate data analysis revealed clear trends in biophysical properties upon PEGylation for a subset of proteins, although no universal trends were found. Taken together, these findings are important in the consideration of biophysical methods and evaluation of second-generation biopharmaceutical drug candidates.
U2 - 10.1021/acs.molpharmaceut.6b00049
DO - 10.1021/acs.molpharmaceut.6b00049
M3 - Journal article
C2 - 27043713
VL - 13
SP - 1587
EP - 1598
JO - Molecular Pharmaceutics
JF - Molecular Pharmaceutics
SN - 1543-8384
IS - 5
ER -