National Institutes for Food and Drug Control, No. 2, Tiantan Xili, Dongcheng District, Beijing 100050, China.
Institutes of Process Engineering, Chinese Academy of Sciences, 1 North 2nd Street, Zhongguancun, Haidian District, Beijing 100190, China.
Molecules. 2019 Feb 15;24(4):699. doi: 10.3390/molecules24040699.
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.
治疗用重组人角质细胞生长因子 1（rhKGF-1）于 2004 年经美国食品和药物管理局（FDA）批准，用于治疗血液系统恶性肿瘤患者造血干细胞移植后引起的口腔黏膜炎。然而，美国药典中并未记录 rhKGF-1 生物活性的推荐生物测定方法。在本研究中，我们开发了一种 rhKGF-1 依赖性生物测定法，用于基于稳定表达受血清反应元件（SRE）和人成纤维细胞生长因子受体 2b（FGFR2b）驱动的荧光素酶报告基因的 HEK293 和 HaCat 细胞系来测定 rhKGF-1 的生物活性。目标 HEK293/HaCat 细胞系对 rhKGF-1 和 rhKGF-2 表现出良好的响应性。我们的严格验证完全按照国际协调会议（ICH）Q2（R1）指南、AAPS/FDA 生物分析研讨会和中国药典的要求，专注于特异性、线性、准确性、精密度和稳健性。我们证实了该方法在测定 rhKGF 生物活性方面的可靠性。验证后的方法省时、灵敏、简单，对于在治疗性 rhKGF 的制造、研究和开发过程中提供质量控制信息特别有价值。
