title: Assessment of the structure of pegylated-recombinant protein therapeutics by the NMR fingerprint assay.
abstract: A number of recombinant protein therapeutic products, such as filgrastim (methionyl granulocyte colony stimulating factor [Met-GCSF] used to boost the immune system in chemotherapy treated cancer patients), and interferon alpha-2 (used for the treatment of various viral infections), have been chemically modified with the addition of a polyethylene glycol (PEG) chain. This modification prolongs residency of the drug in the body and reduces metabolic degradation, which allows less frequent administration of the products. Here we show how NMR spectroscopy methods can assess the higher order structure (HOS) of pegylated-filgrastim (Neulasta®), pegylated interferon-α2a (Pegasys®) pegylated interferon-α2b (PEG-Intron®) purchased from the marketplace. The addition of the PEG moiety effectively doubles the molecular weight of the three products. This presents a significant challenge for the application of NMR techniques. Nevertheless, the results showed that high-resolution spectra could be recorded for two of the three products. Comparison of the spectra of the pegylated protein and the non-pegylated protein shows that the chemical modification did not alter the HOS of these proteins.
authors.affiliation: Centre for Biologics Evaluation, Regulatory Research Division, Biologics and Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa, Ontario K1A 0K9, Canada.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Hodgson
authors.affiliation: Centre for Biologics Evaluation, Regulatory Research Division, Biologics and Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa, Ontario K1A 0K9, Canada; Department of Chemistry, Carleton University, 1125 Colonel By, Ottawa, Ontario K1S 5B6, Canada. Electronic address: yves.aubin@hc-sc.gc.ca.
authors.forename: Yves
authors.initials: Y
authors.lastname: Aubin
mesh_terms: D001681:Biological Assay / Q000379:methods; D000069585:Filgrastim / Q000737:chemistry*; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry; D008279:Magnetic Resonance Imaging / Q000379:methods; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry*
chemical_list: D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; C417083:peginterferon alfa-2b; D000069585:Filgrastim; C100416:peginterferon alfa-2a
keywords: Higher order structure; NMR spectroscopy; Pegylation; Protein structure; Recombinant protein therapeutics