Xie Tao, Ouyang Weiming, Zhang Yaqin, Angart Phillip, Lynch Patrick J, Frucht David M
Office of Pharmaceutical Quality Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
Office of Pharmaceutical Quality Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD 20993, USA.
J Pharm Biomed Anal. 2025 Nov 15;265:117056. doi: 10.1016/j.jpba.2025.117056. Epub 2025 Jul 7.
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.
人粒细胞集落刺激因子（G-CSF）是促进中性粒细胞发育和功能的主要细胞因子。已经开发出十几种重组人G-CSF（rhG-CSF）治疗性原研产品或生物类似药产品（例如非格司亭和培非格司亭），并广泛用于治疗和预防中性粒细胞减少症，尤其是在癌症化疗之后。已报道的用于评估rhG-CSF产品生物活性的分析方法主要基于G-CSF诱导的小鼠骨髓母细胞NSF-60或其变异细胞系的增殖。据报道，这些细胞增殖试验在不同实验室之间的试验性能存在很大差异。此外，由于G-CSF受体存在种间差异，人G-CSF与NFS-60细胞上表达的小鼠G-CSF受体相互作用引发的生物学作用不能完全反映该产品在人体内的作用。我们在此描述了一种新的293-CSF3R-STAT3Luc报告细胞系的建立，该细胞系组成性表达人G-CSF受体，并在响应STAT3激活时诱导表达荧光素酶。该细胞系对rhG-CSF的刺激有选择性反应。利用该细胞系，我们开发并验证了一种基于报告基因的PEG-rhG-CSF产品效价生物测定法。这种新的效价报告基因测定法在参考物质效价的25%-200%范围内呈线性且准确，并且该测定法具有可接受的特异性、精密度和稳健性。这种报告细胞测定平台也可用于开发rhG-CSF类中其他治疗药物效价测定的方法。
