title: Selective functional activity measurement of a PEGylated protein with a modification-dependent activity assay.
abstract: BAX 855 (ADYNOVATE) is a PEGylated recombinant factor VIII (rFVIII) that showed prolonged circulatory half-life compared to unmodified rFVIII in hemophilic patients. Here, the development and validation of a novel assay is described that selectively measures the activity of BAX 855 as cofactor for the serine protease factor IX, which actives factor X. This method type, termed modification-dependent activity assay, is based on PEG-specific capture of BAX 855 by an anti-PEG IgG preparation, followed by a chromogenic FVIII activity assay. The assay principle enabled sensitive measurement of the FVIII cofactor activity of BAX 855 down to the pM-range without interference by non-PEGylated FVIII. The selectivity of the capture step, shown by competition studies to primarily target the terminal methoxy group of PEG, also allowed assessment of the intactness of the attached PEG chains. Altogether, the modification-dependent activity not only enriches, but complements the group of methods to selectively, accurately, and precisely measure a PEGylated drug in complex biological matrices. In contrast to all other methods described so far, it allows measurement of the biological activity of the PEGylated protein. Data obtained demonstrate that this new method principle can be extended to protein modifications other than PEGylation and to a variety of functional activity assays.
authors.affiliation: Shire, Vienna, Austria.
authors.forename: Alfred
authors.initials: A
authors.lastname: Weber
authors.affiliation: Shire, Vienna, Austria.
authors.forename: Andrea
authors.initials: A
authors.lastname: Engelmaier
authors.affiliation: Shire, Vienna, Austria.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Mohr
authors.affiliation: Shire, Vienna, Austria.
authors.forename: Sonja
authors.initials: S
authors.lastname: Haindl
authors.affiliation: Shire, Vienna, Austria.
authors.forename: Hans Peter
authors.initials: HP
authors.lastname: Schwarz
authors.affiliation: Shire, Vienna, Austria. Electronic address: peter.turecek@shire.com.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Turecek
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay; D002138:Calibration; D002352:Carrier Proteins / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D005169:Factor VIII / Q000032:analysis* / Q000737:chemistry; D006207:Half-Life; D007074:Immunoglobulin G / Q000737:chemistry; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D015203:Reproducibility of Results
chemical_list: D002352:Carrier Proteins; C428125:F8a protein, mouse; D007074:Immunoglobulin G; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C000609799:BAX 855; D005169:Factor VIII
keywords: Activity assay; PEGylated drug; Pharmacokinetic