title: N-terminal specificity of PEGylation of human bone morphogenetic protein-2 at acidic pH.
abstract: Site-specific PEGylation offers the possibility to modify a therapeutic protein without interfering with its biological activity. Previously, a preferential N-terminal PEGylation has been reported for several proteins when the reaction was performed at acidic pH. In the present study it was explored if acidic pH favors N-terminal PEGylation of bone morphogenetic protein-2 (BMP-2). PEGylation by poly(ethylene glycol) aldehyde (PEG-AL) or poly(ethylene glycol) carboxymethyl succinimidyl ester (PEG-NHS) was performed at moderate acidic pH of 4. Comparing with PEG-NHS, PEG-AL converted more BMP-2 mainly to mono- or di-PEGylated derivatives at much less molar excess and shorter duration. Analysis of Tryptic fragments of the PEGylated derivatives indicated a partial N-terminal PEGylation specificity. PEG-AL exhibited higher specificity than PEG-NHS. UV spectrometry proved that PEGylation improved the solubility of BMP-2 in PBS. Surface plasmon resonance showed that PEGylation decreased the binding of BMP-2 proteins to a type II receptor. Remarkably, mono-PEGylated BMP-2 with PEG-AL showed higher cellular bioactivity than unmodified protein. Higher N-terminal PEGylation specificity correlates with higher receptor binding affinity and cellular activity. In summary, PEGylation of BMP-2 by PEG-AL and PEG-NHS at acidic pH exhibits a partial N-terminal specificity which however might be sufficient for an efficient site-specific PEGylation process.
authors.affiliation: Physiological Chemistry II, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
authors.forename: Junli
authors.initials: J
authors.lastname: Hu
authors.forename: Walter
authors.initials: W
authors.lastname: Sebald
mesh_terms: D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D015374:Biosensing Techniques; D055396:Bone Morphogenetic Protein 2 / Q000737:chemistry* / Q000502:physiology; D052004:Bone Morphogenetic Protein Receptors / Q000378:metabolism; D002460:Cell Line; D016207:Cytokines / Q000737:chemistry / Q000502:physiology; D004339:Drug Compounding; D016503:Drug Delivery Systems; D005079:Excipients / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008968:Molecular Conformation; D032446:Myoblasts; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D012995:Solubility; D014357:Trypsin / Q000378:metabolism
chemical_list: D000447:Aldehydes; C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D016207:Cytokines; D005079:Excipients; D011506:Proteins; D011092:Polyethylene Glycols; D052004:Bone Morphogenetic Protein Receptors; D014357:Trypsin