Yves Aubin1, Derek J Hodgson2, William B Thach2, Geneviève Gingras2, Simon Sauvé2. 1. 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. 2. 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.
Abstract
PURPOSE: 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.
PURPOSE: Filgrastim is the generic name for recombinant methionyl humangranulocyte 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.
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Authors: Robert G Brinson; John P Marino; Frank Delaglio; Luke W Arbogast; Ryan M Evans; Anthony Kearsley; Geneviève Gingras; Houman Ghasriani; Yves Aubin; Gregory K Pierens; Xinying Jia; Mehdi Mobli; Hamish G Grant; David W Keizer; Kristian Schweimer; Jonas Ståhle; Göran Widmalm; Edward R Zartler; Chad W Lawrence; Patrick N Reardon; John R Cort; Ping Xu; Feng Ni; Saeko Yanaka; Koichi Kato; Stuart R Parnham; Desiree Tsao; Andreas Blomgren; Torgny Rundlöf; Nils Trieloff; Peter Schmieder; Alfred Ross; Ken Skidmore; Kang Chen; David Keire; Darón I Freedberg; Thea Suter-Stahel; Gerhard Wider; Gregor Ilc; Janez Plavec; Scott A Bradley; Donna M Baldisseri; Mauricio Luis Sforça; Ana Carolina de Mattos Zeri; Julie Yu Wei; Christina M Szabo; Carlos A Amezcua; John B Jordan; Mats Wikström Journal: MAbs Date: 2018-12-22 Impact factor: 5.857