title: CHO cell expression, long-term stability, and primate pharmacokinetics and brain uptake of an IgG-paroxonase-1 fusion protein.
abstract: Paraoxonase (PON)-1 is the most potent human organophosphatase known, but recombinant forms of human PON1 have been difficult to produce owing to poor secretion by host cells. In the present investigation, human PON1 is re-engineered as an IgG-PON1 fusion protein. The 355 amino acid human PON1 is fused to the carboxyl terminus of the heavy chain of a chimeric monoclonal antibody (MAb) against the human insulin receptor (HIR), and this fusion protein is designated HIRMAb-PON1. The HIRMAb part of the fusion protein enables brain penetration of the PON1, which was considered important, because organophosphate toxicity causes death via a central nervous system site of action. A high producing line of stably transfected Chinese hamster ovary (CHO) cells secreting the HIRMAb-PON1 fusion protein in the absence of serum or lipid acceptors was cloned. The bioreactor generated fusion protein was purified to homogeneity with low impurities by protein A affinity chromatography and anion exchange chromatography. The HIRMAb-PON1 fusion protein was stable as a sterile liquid formulation stored at 4°C for at least 1 year. The plasma pharmacokinetics (PK) of the HIRMAb-PON1 fusion protein was evaluated in Rhesus monkeys, which is the first PK evaluation of a recombinant PON1 protein. The fusion protein was rapidly removed from blood, primarily by the liver. The blood-brain barrier permeation of the HIRMAb-PON1 fusion protein was high and comparable to other HIRMAb fusion proteins. Re-engineering human PON1 as the HIRMAb fusion protein allows for production of a stable, field-deployable formulation of the enzyme that is brain-penetrating.
authors.affiliation: ArmaGen Technologies, Inc., Santa Monica, California, USA.
authors.forename: Ruben J
authors.initials: RJ
authors.lastname: Boado
authors.forename: Eric Ka-Wai
authors.initials: EK
authors.lastname: Hui
authors.forename: Jeff Zhiqiang
authors.initials: JZ
authors.lastname: Lu
authors.forename: William M
authors.initials: WM
authors.lastname: Pardridge
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000493:pharmacokinetics*; D043303:Aryldialkylphosphatase / Q000235:genetics / Q000493:pharmacokinetics*; D001688:Biological Products / Q000235:genetics / Q000302:isolation & purification / Q000493:pharmacokinetics*; D001812:Blood-Brain Barrier; D001921:Brain / Q000187:drug effects*; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D004355:Drug Stability; D007074:Immunoglobulin G / Q000235:genetics; D008253:Macaca mulatta; D011972:Receptor, Insulin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000493:pharmacokinetics*; D012034:Refrigeration; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D001688:Biological Products; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D011972:Receptor, Insulin; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human