Correlation of liquid chromatographic and biological assay__ for potency assessment of filgrastim an.pdf

C a A M a b a A R R A A K F I C B C 1 b s d a m b c r O r t fl 0 dJournal of Pharmaceutical and Biomedical Analysis 53 (2010) 262–268 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journa l homepage: www.e lsev ier .com/ locate / jpba orrelation of liquid chromatographic and biological assay for potency ssessment of filgrastim and related impurities na Skrlina,?, Ela Kosor Krnica, Darko Gosaka, Berislav Prestera, Vladimir Mrsab, arko Vuletica, Domagoj Runaca Hospira Zagreb d.o.o., Prilaz Baruna Filipovica 27/D, 10 000 Zagreb, Croatia Faculty of Food Technology and Biotechnology, Pierottijeva 6, 10 000 Zagreb, Croatia r t i c l e i n f o rticle history: eceived 5 November 2009 eceived in revised form 2 February 2010 ccepted 4 February 2010 vailable online 12 February 2010 eywords: ilgrastim mpurities hromatography iological assay orrelation a b s t r a c t In vivo and in vitro potency assays have always been a critical tool for confirmation of protein activity. However, due to their complexity and time consuming procedures, it remains a challenge to find an alter- native analytical approach thatwould enable their replacementwith no impact on the quality of provided information. The goal of this research was to determine if a correlation between liquid chromatography assays and in vitro biological assay could be established for filgrastim (recombinant human granulocyte- colony stimulating factor, rhG-CSF) samples containing various amounts of related impurities. For that purpose, relevant filgrastim related impurities were purified to homogeneity and characterized by liquid chromatography and mass spectrometry. A significant correlation (R2 0.90) between the two types of assays was revealed. Potency of oxidized filgrastim was determined to be approximately 25% of filgras- tim stated potency (1×108 IU/mg of protein). Formyl-methionine filgrastim had potency of 89% of the filgrastim stated potency, while filgrastim dimer had