title: Large-Scale Biophysical Evaluation of Protein PEGylation Effects: In Vitro Properties of 61 Protein Entities.
abstract: 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.
authors.affiliation: Novo Nordisk A/S , 2880 Bagsværd, Denmark.|The Novo Nordisk Foundation Center for Protein Research (NNF CPR), Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Erik
authors.initials: E
authors.lastname: Vernet
authors.affiliation: The Novo Nordisk Foundation Center for Protein Research (NNF CPR), Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Gina
authors.initials: G
authors.lastname: Popa
authors.affiliation: The Novo Nordisk Foundation Center for Protein Research (NNF CPR), Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Irina
authors.initials: I
authors.lastname: Pozdnyakova
authors.affiliation: Department of Chemistry, Faculty of Science, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Jakob E
authors.initials: JE
authors.lastname: Rasmussen
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Holger
authors.initials: H
authors.lastname: Grohganz
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Lise
authors.initials: L
authors.lastname: Giehm
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Malene H
authors.initials: MH
authors.lastname: Jensen
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Huabing
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Bitten
authors.initials: B
authors.lastname: Plesner
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Hanne M
authors.initials: HM
authors.lastname: Nielsen
authors.affiliation: Department of Chemistry, Faculty of Science, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
authors.affiliation: Costerton Biofilm center, Department for Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Jens
authors.initials: J
authors.lastname: Berthelsen
authors.affiliation: The Novo Nordisk Foundation Center for Protein Research (NNF CPR), Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Michael
authors.initials: M
authors.lastname: Sundström
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen, Denmark.
authors.forename: Marco
authors.initials: M
authors.lastname: van de Weert
mesh_terms: D001703:Biophysics / Q000379:methods; D002152:Calorimetry, Differential Scanning / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002942:Circular Dichroism / Q000379:methods; D011092:Polyethylene Glycols / Q000737:chemistry*; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry*; D013696:Temperature
chemical_list: D011506:Proteins; D011092:Polyethylene Glycols
keywords: PEGylation; analytical chemistry; biopharmaceuticals characterization; circular dichroism; conjugation; dynamic light scattering; multivariate analysis; physicochemical properties; principal component analysis; proteins