title: Tungsten-induced denaturation and aggregation of epoetin alfa during primary packaging as a cause of immunogenicity.
abstract: OBJECTIVE
  Following two cases of neutralizing antibodies to epoetin alfa in an investigational clinical study, a small number of individual syringes of two drug product batches were found to contain unusually high levels of aggregation at the end of the clinical trial.
  
  
  METHODS
  We undertook an extensive analytical approach to determine the root-cause of the increased aggregation in the affected batches.
  
  
  RESULTS
  Soluble tungsten was found in the syringes, most likely derived from the pins used to manufacture the syringes. Spiking of epoetin alfa with sodium polytungstate or an extract of tungsten pins used to manufacture the syringes induced the formation of aggregates, both dimers that appeared to be covalently linked by disulphide bonds as well as higher-order aggregates. Sodium polytungstate had also a strong denaturing effect on the protein.
  
  
  CONCLUSIONS
  We propose tungsten-mediated unfolding and aggregation of epoetin alfa in pre-filled syringes as a potential root cause for increased immunogenicity. This finding may be more broadly applicable to this and other classes of therapeutic proteins.
authors.affiliation: Sandoz Biopharmaceuticals, Hexal AG, Keltenring 1 + 3, 82041, Oberhaching, Germany. andreas.seidl@sandoz.com
authors.forename: Andreas
authors.initials: A
authors.lastname: Seidl
authors.forename: Otmar
authors.initials: O
authors.lastname: Hainzl
authors.forename: Marleen
authors.initials: M
authors.lastname: Richter
authors.forename: Robert
authors.initials: R
authors.lastname: Fischer
authors.forename: Stephan
authors.initials: S
authors.lastname: Böhm
authors.forename: Britta
authors.initials: B
authors.lastname: Deutel
authors.forename: Martin
authors.initials: M
authors.lastname: Hartinger
authors.forename: Jörg
authors.initials: J
authors.lastname: Windisch
authors.forename: Nicole
authors.initials: N
authors.lastname: Casadevall
authors.forename: Gerard Michel
authors.initials: GM
authors.lastname: London
authors.forename: Iain
authors.initials: I
authors.lastname: Macdougall
mesh_terms: D057134:Antibodies, Neutralizing / Q000097:blood*; D002626:Chemistry, Pharmaceutical; D004339:Drug Compounding; D004340:Drug Contamination; D004349:Drug Packaging; D004355:Drug Stability; D000068817:Epoetin Alfa; D004921:Erythropoietin / Q000737:chemistry / Q000276:immunology*; D006397:Hematinics / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D011489:Protein Denaturation; D055503:Protein Multimerization; D058767:Protein Unfolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D013594:Syringes; D013678:Technology, Pharmaceutical / Q000379:methods; D014414:Tungsten / Q000009:adverse effects* / Q000737:chemistry
chemical_list: D057134:Antibodies, Neutralizing; D006397:Hematinics; D011994:Recombinant Proteins; D004921:Erythropoietin; D000068817:Epoetin Alfa; D014414:Tungsten
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