Structure−Function Engineering of Interferon-β-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.

PEGylation of therapeutic proteins

“…Significantly improved physico-chemical characteristics after coupling of PEG to a protein can be explained by the increased hydrodynamic volume of the PEG-protein conjugate resulting from the ability of PEG to coordinate water molecules and from the high flexibility of the PEG chain. Consequently, apparent Mw of the PEG-protein conjugate is around 5 to 10 fold higher in comparison to the globular protein of the same nominal Mw [26]. PEG chains can sweep around the protein to shield and protect it from the environment (or vice versa), but they also influence the interactions of the protein that are responsible for its biological function.…”

In situ growth of a stoichiometric PEG-like conjugate at a protein's N-terminus with significantly improved pharmacokinetics

“…Modification of lysine or cysteine residues of proteins with poly(ethylene glycol) (PEG), generally known as PEGylation, is the most widely used polymer conjugation approach to improve the pharmacological profiles of proteins, and several PEGylated proteins are used clinically as therapeutics (2)(3)(4)(5). Unfortunately, most proteins contain numerous chemically reactive residues that are promiscuously distributed on their surface, which can result in the formation of heterogeneous protein-polymer conjugates with unacceptably low biological activity (6)(7)(8)(9).…”

Artificial Blood- A Game Changer for Future Medicine: Where are we Today?

“…For instance, recent studies reported that covalent attachments of poly(ethylene) glycol to the surfaces of acetylcholinesterase or interferon-β-1b have shown a meaningful increase in the circulatory residency times [5,6]. Similarly, conjugation of poly(ethylene) glycol with hemoglobin has been suggested to act a hemoglobin-based oxygen carrier.…”