A new STAT3-based potency assay for human G-CSF analog therapeutics.

Human granulocyte colony-stimulating factor (G-CSF) is the primary cytokine promoting the development and function of neutrophils. More than a dozen recombinant human G-CSF (rhG-CSF) therapeutic originator or biosimilar products (e.g., filgrastim and pegfilgrastim) have been developed and are widely used to treat and prevent neutropenia, especially following chemotherapy for cancer. Published analytical methods described for assessing the bioactivities of rhG-CSF products are primarily based on G-CSF-induced proliferation of the murine myeloblastic NSF-60 or its variant cell lines. These cell proliferation assays are reported to exhibit large variability in assay performance between laboratories. Moreover, the biological action initiated by the interaction of human G-CSF with the murine G-CSF receptor expressed on NFS-60 cells does not fully reflect the action of the product in humans because of interspecies differences between the G-CSF receptors. We describe herein the establishment of a new 293-CSF3R-STAT3Luc reporter cell line that constitutively expressed the human G-CSF receptor and inducibly expressed luciferase in response to STAT3 activation. This cell line selectively responded to stimulation by rhG-CSF. Using this cell line, we developed and qualified a reporter-based potency bioassay for PEG-rhG-CSF products. This new potency reporter assay was linear and accurate over the range of 25-200 % of the reference material potency, and the assay demonstrated acceptable specificity, precision, and robustness. This reporter cell assay platform may also be applied to develop assays for potency determinations of other therapeutics in the rhG-CSF class.