Tungsten-Induced Denaturation and Aggregation of Epoetin Alfa During Primary Packaging as a Cause of Immunogenicity

• Published in: Pharmaceutical research Vol. 29; no. 6; pp. 1454 - 1467
• Main Authors: Seidl, Andreas, Hainzl, Otmar, Richter, Marleen, Fischer, Robert, Böhm, Stephan, Deutel, Britta, Hartinger, Martin, Windisch, Jörg, Casadevall, Nicole, London, Gerard Michel, Macdougall, Iain
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01.06.2012; Springer; Springer Nature B.V
• Subjects: Antibodies, Neutralizing - blood; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering and Bioengineering; Biomedicine; Chemistry, Pharmaceutical; Drug Compounding; Drug Contamination; Drug Packaging; Drug Stability; Epoetin Alfa; Erythropoietin - chemistry; Erythropoietin - immunology; General pharmacology; Hematinics - chemistry; Hematinics - immunology; Humans; Immunoglobulins; Medical Law; Medical sciences; Pharmaceutical technology. Pharmaceutical industry; Pharmacology; Pharmacology. Drug treatments; Pharmacology/Toxicology; Pharmacy; Protein Denaturation; Protein Multimerization; Protein Unfolding; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - immunology; Research Paper; Syringes; Technology, Pharmaceutical - methods; Tungsten - adverse effects; Tungsten - chemistry; epoetin; aggregation; tungsten; Aggregation; Pharmaceutical technology; Immunogenicity; Denaturation; Packaging; Epoetin alfa; Antianemia agent
• ISSN: 0724-8741; 1573-904X
• DOI: 10.1007/s11095-011-0621-4

ABSTRACT Purpose 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.