title: Monitoring Effects of Excipients, Formulation Parameters and Mutations on the High Order Structure of Filgrastim by NMR.
abstract: OBJECTIVE
  Filgrastim is the generic name for recombinant methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF). It is marketed under the brand name Neupogen® by Amgen. Since this product has lost patent protection, many biosimilar versions have been approved or are in the process of filing for market authorization throughout the world. Here we show that NMR spectroscopy can be used to assess the three-dimensional structure of the active ingredient in the formulated approved product Neupogen®.
  
  
  METHODS
  Recombinant metHuG-CSF was prepared in E. coli and isotopically enriched with (13)C and (15) N isotopes. NMR spectroscopy was used to study the effects of excipients on the conformation.
  
  
  RESULTS
  The effects of pH variation on the amide chemical shifts suggest the presence of cation-pi interactions between His-79 and Trp-118, and His-156-Trp-58-His-52 that stabilizes the conformation at low pH. This may be associated with a small local conformational change. The NMR data showed that polysorbate does not interact significantly with filgrastim thus allowing the collection of spectra in the presence of 20 times the formulation concentration in the sample. However, at higher detergent concentrations a reduction of signal intensity is observed. Conclusions The NMR fingerprint assay applied to filgrastim (Neupogen® and a CRS from the European Pharmacopeia (EP)) provided residue specific information of the structure of the drug substance. In addition to current methods, the ability to assess the conformation with a high degree of resolution can greatly facilitate comparability exercises.
authors.affiliation: Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Tunney's Pasture, A/L 2201E, Ottawa, Ontario, K1A 0K9, Canada. yves.aubin@hc-sc.gc.ca.
authors.forename: Yves
authors.identifier: http://orcid.org/0000-0002-8554-0297
authors.initials: Y
authors.lastname: Aubin
authors.affiliation: Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Tunney's Pasture, A/L 2201E, Ottawa, Ontario, K1A 0K9, Canada.
authors.forename: Derek J
authors.initials: DJ
authors.lastname: Hodgson
authors.affiliation: Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Tunney's Pasture, A/L 2201E, Ottawa, Ontario, K1A 0K9, Canada.
authors.forename: William B
authors.initials: WB
authors.lastname: Thach
authors.affiliation: Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Tunney's Pasture, A/L 2201E, Ottawa, Ontario, K1A 0K9, Canada.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Gingras
authors.affiliation: Centre for Biologics Evaluation, Biologics and Genetic Therapies Directorate, Health Canada, 251 Sir Frederick Banting Driveway, Tunney's Pasture, A/L 2201E, Ottawa, Ontario, K1A 0K9, Canada.
authors.forename: Simon
authors.initials: S
authors.lastname: Sauvé
mesh_terms: D002626:Chemistry, Pharmaceutical / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005079:Excipients / Q000737:chemistry*; D000069585:Filgrastim / Q000737:chemistry* / Q000235:genetics*; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008279:Magnetic Resonance Imaging / Q000379:methods; D009154:Mutation / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D005079:Excipients; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D000069585:Filgrastim
keywords: NMR; excipients; filgrastim; formulation; structure