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J Immunol Methods. 2013 Oct 31;396(1-2):1-7. doi: 10.1016/j.jim.2013.07.007. Epub 2013 Aug 6.
The quality of bioanalytical data is dependent upon selective, sensitive, and reproducible analytical methods. With evolving technologies available, bioanalytical scientists must assess which is most appropriate for their molecule through proper method validation. For an early stage PEGylated insulin program, the characteristics of four platforms, ELISA, ECL, Gyrolab, and LC-MS/MS, were evaluated using fit-for-purpose method development and validation, while also evaluating costs.
Methods selected for validation required acceptable performance based on satisfaction of a priori criteria prior to proceeding to subsequent stages of validation. LBA pre-validation included reagent selection, evaluation of matrix interference, and range determination. LC-MS/MS pre-validation included selection of a signature peptide; optimization of sample preparation, HPLC, and LC-MS/MS conditions; and calibration range determination. Pre-study validation tested accuracy and precision (mean bias criteria±30%; precision≤30%). Pharmacokinetic (PK) parameters were estimated for an in vivo study with WinNonlin noncompartmental analysis. Statistics were performed with JMP using ANOVA and Tukey-Kramer post hoc analysis. A cost analysis was performed for a 200-sample PK study using the methods from this study.
All platforms, except Gyrolab, were taken through validation. However, a typical Gyrolab method was included for the cost analysis. Ranges for the ELISA, ECLA, and LC-MS/MS were 8.52-75, 2.09-125, and 100-1000 ng/mL, respectively, and accuracy and precision fell within a priori criteria. PK samples were analyzed in the 3 validated methods. PK profiles and parameters are similar for all methods, except LC-MS/MS, which differed at t=24h and with AUC0-24. Further investigation into this difference is warranted. The cost analysis identified the Gyrolab platform as the most expensive and ELISA as the least expensive, with method specific consumables attributing significantly to costs.
ECLA had a larger dynamic range and sensitivity, allowing accurate assessment of PK parameters. Although this method was more expensive than the ELISA, it was the most appropriate for the early stage PEGylated insulin program. While this case study is specific to PEGylated human insulin, it highlights the importance of evaluating and selecting the most appropriate platform for bioanalysis during drug development.
生物分析数据的质量取决于选择性、灵敏性和重现性分析方法。随着现有技术的发展，生物分析科学家必须通过适当的方法验证来评估哪种方法最适合他们的分子。在一个早期的聚乙二醇化胰岛素项目中，使用适合目的的方法开发和验证评估了四种平台（ELISA、ECL、Gyrolab 和 LC-MS/MS）的特性，同时还评估了成本。
为了进行验证，选择的方法需要基于满足先验标准的可接受性能，然后才能进行验证的后续阶段。LBA 预验证包括试剂选择、基质干扰评估和范围确定。LC-MS/MS 预验证包括选择特征肽；优化样品制备、HPLC 和 LC-MS/MS 条件；以及校准范围确定。预研究验证测试了准确度和精密度（平均偏差标准±30%；精密度≤30%）。使用 WinNonlin 非房室分析估算体内研究的药代动力学（PK）参数。使用 JMP 通过 ANOVA 和 Tukey-Kramer 事后分析进行统计分析。使用本研究中的方法对 200 个 PK 研究样本进行了成本分析。
除 Gyrolab 外，所有平台都经过了验证。然而，为了进行成本分析，包括了一个典型的 Gyrolab 方法。ELISA、ECLA 和 LC-MS/MS 的范围分别为 8.52-75、2.09-125 和 100-1000ng/mL，准确度和精密度均符合先验标准。在 3 种经过验证的方法中分析了 PK 样品。除 LC-MS/MS 外，所有方法的 PK 曲线和参数均相似，LC-MS/MS 在 t=24h 时和 AUC0-24 时存在差异。需要进一步研究这种差异。成本分析表明 Gyrolab 平台成本最高，ELISA 平台成本最低，方法专用耗材对成本有显著影响。
ECLA 具有更大的动态范围和灵敏度，能够准确评估 PK 参数。虽然这种方法比 ELISA 更昂贵，但它最适合早期的聚乙二醇化胰岛素项目。虽然这个案例研究是针对聚乙二醇化人胰岛素的，但它强调了在药物开发过程中评估和选择最适合生物分析的平台的重要性。
