title: Hepatocyte clearance and pharmacokinetics of recombinant factor IX glycosylation variants.
abstract: Addition of N-linked glycosylation sites has been shown to increase serum half-life and decrease clearance for proteins such as recombinant erythropoietin (EPO). However, factor IX (FIX) variants with additional N-linked glycans ("HG" variants) that were expressed in HKB11 cells showed increased clearance in rat in vivo pharmacokinetic studies relative to FIX variants with no additional glycans. Variants with multiple additional glycans were the most rapidly cleared. A rat hepatocyte clearance assay was developed to measure intrinsic clearance of these FIX variants in vitro. The rank order of clearance of the variants was the same both in vivo and in the in vitro hepatocyte assay. In the in vitro assay, heparin, galactose, and asialo-orosomucoid inhibited clearance of a FIX HG variant by hepatocytes, and asialo-FIX was rapidly cleared, suggesting roles for the asialoglycoprotein receptor (ASGPR) and cell surface proteoglycans in FIX clearance. Thus the in vitro hepatocyte intrinsic clearance assay is both useful and predictive for identifying rapidly cleared recombinant proteins and for helping to identify receptors involved in clearance of proteins by the liver.
authors.affiliation: Bayer Healthcare Pharmaceuticals, 455 Mission Bay Blvd South, San Francisco, CA, USA. Electronic address: eric.blasko@bayer.com.
authors.forename: Eric
authors.initials: E
authors.lastname: Blasko
authors.forename: Alan R
authors.initials: AR
authors.lastname: Brooks
authors.forename: Elena
authors.initials: E
authors.lastname: Ho
authors.forename: James M
authors.initials: JM
authors.lastname: Wu
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Zhao
authors.forename: Babu
authors.initials: B
authors.lastname: Subramanyam
mesh_terms: D000818:Animals; D002460:Cell Line; D005164:Factor IX / Q000737:chemistry / Q000493:pharmacokinetics*; D006031:Glycosylation; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D008297:Male; D008657:Metabolic Clearance Rate; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000493:pharmacokinetics*
chemical_list: D011994:Recombinant Proteins; D005164:Factor IX; D019158:N-Acetylneuraminic Acid
keywords: Asialoglycoprotein receptor (ASGPR); Clearance; Coagulation; Factor IX; Hepatocyte