title: Identification of Low-Level Product-Related Variants in Filgrastim Products Presently Available in Highly Regulated Markets.
abstract: BACKGROUND
  Filgrastim is a recombinant, non-glycosylated form of human granulocyte colony-stimulating factor, used to stimulate leukocyte proliferation in patients suffering from neutropenia. Since the expiration of patents associated with Amgen's filgrastim biopharmaceutical, Neupogen(®), in 2006, a number of filgrastim products have been marketed; however, a detailed characterization and comparison of variants associated with these products have not been publically reported.
  
  
  OBJECTIVE
  The objective of this study was to identify and quantify product-related variants in filgrastim reference products and biosimilars thereof that are presently available in highly regulated markets.
  
  
  METHODS
  In this study, we used intact and top-down mass spectrometry to identify and quantify product-related variants in filgrastim products. Mass spectrometry has become the method of choice for physicochemical characterization of biopharmaceuticals, allowing accurate and sensitive characterization of product-related variants.
  
  
  RESULTS
  In addition to modifications ubiquitously present in biopharmaceuticals, such as methionine oxidation and asparagine/glutamine deamidation, we identified six different low-level, product-related variants present in some, but not all, of the tested products. Two variants, an acetylated filgrastim variant and a filgrastim variant containing an additional C-terminal tryptophan extension, are newly identified variants.
  
  
  CONCLUSIONS
  This study demonstrates that filgrastim products already in widespread clinical use in highly regulated markets differ in low-level, product-related variants present at levels mostly below 1 % relative abundance. This study provides a comprehensive catalog of minor differences between filgrastim products and suggests that the filgrastim product-related variants described here are not clinically relevant when present at low abundance.
authors.affiliation: Sandoz Biopharmaceuticals, Sandoz GmbH, Biochemiestraße 10, 6250, Kundl, Austria.
authors.forename: Anna
authors.initials: A
authors.lastname: Hausberger
authors.affiliation: Sandoz Biopharmaceuticals, Sandoz GmbH, Biochemiestraße 10, 6250, Kundl, Austria.
authors.forename: William C
authors.initials: WC
authors.lastname: Lamanna
authors.affiliation: Sandoz Biopharmaceuticals, Hexal AG, Keltenring 1+3, 82041, Oberhaching, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Hartinger
authors.affiliation: Sandoz Biopharmaceuticals, Hexal AG, Keltenring 1+3, 82041, Oberhaching, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Seidl
authors.affiliation: Sandoz Biopharmaceuticals, Sandoz GmbH, Biochemiestraße 10, 6250, Kundl, Austria.
authors.forename: Hansjoerg
authors.initials: H
authors.lastname: Toll
authors.affiliation: Sandoz Biopharmaceuticals, Sandoz GmbH, Biochemiestraße 10, 6250, Kundl, Austria. johann.holzmann@sandoz.com.
authors.forename: Johann
authors.identifier: http://orcid.org/0000-0003-2622-4072
authors.initials: J
authors.lastname: Holzmann
mesh_terms: D059451:Biosimilar Pharmaceuticals / Q000032:analysis* / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D000069585:Filgrastim / Q000032:analysis* / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*
chemical_list: D059451:Biosimilar Pharmaceuticals; D000069585:Filgrastim