title: Structure-function engineering of interferon-beta-1b for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective mono-PEGylation.
abstract: Recombinant interferon-beta-1b (IFN-beta-1b) is used clinically in the treatment of multiple sclerosis. In common with many biological ligands, IFN-beta-1b exhibits a relatively short serum half-life, and bioavailability may be further diminished by neutralizing antibodies. While PEGylation is an approach commonly employed to increase the blood residency time of protein therapeutics, there is a further requisite for molecular engineering approaches to also address the stability, solubility, aggregation, immunogenicity and in vivo exposure of therapeutic proteins. We investigated these five parameters of recombinant human IFN-beta-1b in over 20 site-selective mono-PEGylated or multi-PEGylated IFN-beta-1b bioconjugates. Primary amines were modified by single or multiple attachments of poly(ethylene glycol), either site-specifically at the N-terminus, or randomly on the 11 lysines. In two alternate approaches, site-directed mutagenesis was independently employed in the construction of designed IFN-beta-1b variants containing either a single free cysteine or lysine for site-specific PEGylation. Optimization of conjugate preparation with 12 kDa, 20 kDa, 30 kDa, and 40 kDa amine-selective PEG polymers was achieved, and a comparison of the structural and functional properties of the IFN-beta-1b proteins and their PEGylated counterparts was conducted. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the attachment sites of the PEG polymer. Independent biochemical and bioactivity analyses, including antiviral and antiproliferation bioassays, circular dichroism, capillary electrophoresis, flow cytometric profiling, reversed phase and size exclusion HPLC, and immunoassays demonstrated that the functional activities of the designed IFN-beta-1b conjugates were maintained, while the formation of soluble or insoluble aggregates of IFN-beta-1b was ameliorated. Immunogenicity and pharmacokinetic studies of selected PEGylated IFN-beta-1b compounds in mice and rats demonstrated both diminished IgG responses, and over 100-fold expanded AUC exposure relative to the unmodified protein. The results demonstrate the capacity of this macromolecular engineering strategy to address both pharmacological and formulation challenges for a highly hydrophobic, aggregation-prone protein. The properties of a lead mono-PEGylated candidate, 40 kDa PEG2-IFN-beta-1b, were further investigated in formulation optimization and biological studies.
authors.affiliation: Enzon Pharmaceuticals, 20 Kingsbridge Road, Piscataway, New Jersey 08854-3969, USA.
authors.forename: Amartya
authors.initials: A
authors.lastname: Basu
authors.forename: Karen
authors.initials: K
authors.lastname: Yang
authors.forename: Maoliang
authors.initials: M
authors.lastname: Wang
authors.forename: Sam
authors.initials: S
authors.lastname: Liu
authors.forename: Ramesh
authors.initials: R
authors.lastname: Chintala
authors.forename: Thomas
authors.initials: T
authors.lastname: Palm
authors.forename: Hong
authors.initials: H
authors.lastname: Zhao
authors.forename: Ping
authors.initials: P
authors.lastname: Peng
authors.forename: Dechun
authors.initials: D
authors.lastname: Wu
authors.forename: Zhenfan
authors.initials: Z
authors.lastname: Zhang
authors.forename: Jack
authors.initials: J
authors.lastname: Hua
authors.forename: Ming-Ching
authors.initials: MC
authors.lastname: Hsieh
authors.forename: John
authors.initials: J
authors.lastname: Zhou
authors.forename: Gerald
authors.initials: G
authors.lastname: Petti
authors.forename: Xiguang
authors.initials: X
authors.lastname: Li
authors.forename: Ahsen
authors.initials: A
authors.lastname: Janjua
authors.forename: Magda
authors.initials: M
authors.lastname: Mendez
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Clifford
authors.initials: C
authors.lastname: Longley
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Zhang
authors.forename: Mary
authors.initials: M
authors.lastname: Mehlig
authors.forename: Virna
authors.initials: V
authors.lastname: Borowski
authors.forename: Manickam
authors.initials: M
authors.lastname: Viswanathan
authors.forename: David
authors.initials: D
authors.lastname: Filpula
mesh_terms: D000577:Amides / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000068576:Interferon beta-1b; D016899:Interferon-beta / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism* / Q000493:pharmacokinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry*; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism / Q000493:pharmacokinetics; D012995:Solubility
chemical_list: D000577:Amides; D011994:Recombinant Proteins; D000068576:Interferon beta-1b; D011092:Polyethylene Glycols; D016899:Interferon-beta