Analytical techniques and bioactivity assays to compare the structure and function of filgrastim (granulocyte-colony stimulating factor) therapeutics from different manufacturers
The FDA has approved more than 100 protein and peptide drugs with hundreds more in the pipeline (Lanthier et al. in Nat Rev Drug Discov 7(9):733–737, 2008). Many of these originator biologic products are now coming off patent and are being manufactured by alternate methods than the innovator as follow-on drugs. Because changes to the production method often lead to subtle differences (e.g., degradation products, different posttranslational modifications or impurities) in the therapeutic (Schiestl et al. in Nat Biotechnol 29(4):310–312, 2011), there is a critical need to define techniques to test and insure the quality of these drugs. In addition, the emergence of protein therapeutics manufactured by unapproved methodologies presents an ongoing and growing regulatory challenge. In this work, high-resolution mass spectrometry was used to determine the presence or absence of posttranslational modifications for one FDA-approved and three foreign-sourced, unapproved filgrastim products. Circular dichroism (CD) was used to compare the secondary structure and probe the temperature stability of these products. Native 2D 1H,15N-heteronuclear singular quantum coherence (HSQC) NMR test was applied to these samples to compare the higher-order structure of the four products. Finally, a cell proliferation assay was performed on the filgrastims to compare their bioactivity, and stressed filgrastim was tested in the bioassay to better understand the effects of changes in protein structure on activity. The results showed that orthogonal approaches are capable of characterizing the physiochemical properties of this protein drug and assessing the impact of structural changes on filgrastim purity and potency.
KeywordsTop–down mass spectrometry Granulocyte-colony stimulating factor Bioassay Cell proliferation Circular dichroism NMR
- 4.Nemunaitis J, Rabinowe SN, Singer JW, Bierman PJ, Vose JM, Freedman AS, Onetto N, Gillis S, Oette D, Gold M et al (1991) Recombinant granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for lymphoid cancer. N Engl J Med 324(25):1773–1778CrossRefGoogle Scholar
- 11.Bristow AF, Bird C, Bolgiano B, Thorpe R (2012) Regulatory requirements for therapeutic proteins: the relationship between the conformation and biological activity of filgrastim. Pharmeur Bio Sci Notes 2012:103–117Google Scholar
- 17.Klick S, Muijselaar PG, Waterval J, Eichinger T, Kom C, Gerding TK, Debets AJ, Sanger-van de Griend C, van den Beld C, Somsen GW, De Jong GJ (2005) Toward a generic approach for stress testing of drug substances and drug products. Pharm Tech 29:48–66Google Scholar
- 18.Quali EED, EDQM (2008) European Pharmacopoeia supplement 6.3: supplement 6.3 W/6.4 & 6.5 when available. Worldwide Book ServiceGoogle Scholar