Roswall E C, Mukku V R, Chen A B, Hoff E H, Chu H, McKay P A, Olson K C, Battersby J E, Gehant R L, Meunier A, Garnick R L
Genentech, Inc., South San Francisco, CA 94080-4990, USA.
Biologicals. 1996 Mar;24(1):25-39. doi: 10.1006/biol.1996.0003.
Two methods, High-Performance Receptor Binding Chromatography (HPRBC) and Cell Proliferation (CP), have been developed as alternatives to the classical hypophysectomized rat weight gain bioassay for the determination of potency for recombinant human growth hormone (rhGH). In the HPRBC assay, rhGH is combined with an excess of the soluble extracellular domain of the recombinant human growth hormone receptor (referred to as 'receptor' in the discussion of the HPRBC assay). Nondenaturing size-exclusion chromatography is used to analyzed the resulting complex, which forms in a 2:1 receptor to rhGH ratio. The 2:1 complex is assayed at a concentration near the Kd (approximately 0.4 nM), providing high specificity for rhGH and detection of rhGH variants with reduced activity. In the CP assay, a mouse myeloid leukaemia cell line (FDC-P1) transfected with the full-length receptor is exposed to varying levels of rhGH for 16-20 h. The incorporation of 3H-thymidine into DNA is used as an index of cell proliferation. The results show that the HPRBC assay provides significantly improved precision with a relative standard deviation (RSD) of < or = 5% vs. an RSD of 23% for the rat bioassay. The CP assay has RSDs of 4-16%. Analysis of rhGH variants and mutants shows that the potencies measured by both the HPRBC and CP assays are in general agreement with the rat weight gain bioassay. Both of the HPRBC and CP assays are sufficiently rugged for operating in a Good Manufacturing Practices (GMP) routine batch release testing environment. In vitro alternatives such as the HPRBC and CP assays build a foundation for replacing the hypophysectomized rat weight gain bioassay by correlating receptor dimerization, binding specificity and signal transduction with the biological activity of rhGH.
已开发出两种方法，即高效受体结合色谱法（HPRBC）和细胞增殖法（CP），作为经典的垂体切除大鼠体重增加生物测定法的替代方法，用于测定重组人生长激素（rhGH）的效价。在HPRBC测定中，rhGH与过量的重组人生长激素受体的可溶性细胞外结构域（在HPRBC测定讨论中称为“受体”）结合。使用非变性尺寸排阻色谱法分析所得复合物，该复合物以受体与rhGH 2:1的比例形成。在接近解离常数（约0.4 nM）的浓度下测定2:1复合物，对rhGH具有高特异性，并能检测活性降低的rhGH变体。在CP测定中，用全长受体转染的小鼠髓样白血病细胞系（FDC-P1）暴露于不同水平的rhGH 16 - 20小时。将3H-胸腺嘧啶掺入DNA用作细胞增殖的指标。结果表明，HPRBC测定法的精密度显著提高，相对标准偏差（RSD）≤5%，而大鼠生物测定法的RSD为23%。CP测定法的RSD为4 - 16%。对rhGH变体和突变体的分析表明，HPRBC和CP测定法测得的效价与大鼠体重增加生物测定法总体一致。HPRBC和CP测定法在良好生产规范（GMP）常规批量放行测试环境中操作都足够耐用。诸如HPRBC和CP测定法等体外替代方法通过将受体二聚化、结合特异性和信号转导与rhGH的生物活性相关联，为取代垂体切除大鼠体重增加生物测定法奠定了基础。
