Abstract
The therapeutic recombinant human keratinocyte growth factor 1 (rhKGF-1) was approved by the FDA for oral mucositis resulting from hematopoietic stem cell transplantation for hematological malignancies in 2004. However, no recommended bioassay for rhKGF-1 bioactivity has been recorded in the U.S. Pharmacopoeia. In this study, we developed an rhKGF-1-dependent bioassay for determining rhKGF-1 bioactivity based on HEK293 and HaCat cell lines that stably expressed the luciferase reporter driven by the serum response element (SRE) and human fibroblast growth factor receptor (FGFR2) IIIb. A good responsiveness to rhKGF-1 and rhKGF-2 shared by target HEK293/HaCat cell lines was demonstrated. Our stringent validation was completely focused on specificity, linearity, accuracy, precision, and robustness according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, AAPS/FDA Bioanalytical Workshop and the Chinese Pharmacopoeia. We confirmed the reliability of the method in determining rhKGF bioactivity. The validated method is highly timesaving, sensitive, and simple, and is especially valuable for providing information for quality control during the manufacture, research, and development of therapeutic rhKGF.
Highlights
Keratinocyte growth factor (KGF) was originally isolated from human embryonic lung fibroblast-conditioned medium, and it is a member of the fibroblast growth factor (FGF) family [1,2].The FGF family is composed of 23 members and classified into six subfamilies in mammals [3]
The results demonstrated that HEK293-Luc cells responded to recombinant human keratinocyte growth factor 1 (rhKGF-1) in a dose-dependent not to recombinant human erythropoietin (rhEPO), and nerve growth factor (NGF).and recombinant human epidermal growth factor (rhEGF), a slight appeared, but the relative luciferase units (RLU)
We describe a new bioassay to determine the bioactivity of rhKGF-1 based on the luciferase reporter gene driven by serum response element (SRE) in HEK293 and HaCat cells bearing FGFR2 IIIb
Summary
Keratinocyte growth factor (KGF) was originally isolated from human embryonic lung fibroblast-conditioned medium, and it is a member of the fibroblast growth factor (FGF) family [1,2]. KGF-1 and KGF-2 are considered paracrine factors, and regulate embryonic development by binding to their specific FGF receptor 1 (FGFR1) and FGFR2. Through its intracellular signal transduction, KGF induces its biological activity in keratinocytes and in the development and morphogenesis of multiple epithelial cell lineages within the skin, lung, and reproductive tract [19,20]. An RGA for determining the bioactivity of therapeutic rhKGF was developed based on HEK293 and HaCat cell lines stably transfected with luciferase reporter gene controlled by the serum response element (SRE) promoter and human FGFR2 IIIb. Upon rhKGF binding to FGFR2 IIIb and subsequent intracellular signaling cascades, the interaction between the transcription factor and SRE drives downstream luciferase gene expression. Clones 1C1 and 1A6 were selected for further method validation
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