Department of Protein Analytical Chemistry, Genentech, a Member of the Roche Group, 1 DNA Way, South San Francisco, California 94080, USA.
Anal Chem. 2012 Jun 19;84(12):5380-6. doi: 10.1021/ac3008847. Epub 2012 Jun 5.
Characterization of charge heterogeneity of recombinant monoclonal antibodies (mAbs) requires high throughput analytical methods to support clone selection and formulation screens. We applied the NanoPro technology to rapidly measure relative charge distribution of mAbs in early stage process development. The NanoPro is a multiplexed capillary-based isoelectric immunoassay with whole-column imaging detection. This assay offers specificity, speed and sensitivity advantages over conventional capillary isoelectric focusing (CIEF) platforms. After CIEF, charge variants are photochemically immobilized to the wall of a short coated capillary. Once immobilized, mAbs are probed using a secondary anti-IgG conjugated with horseradish peroxidase. After flushing away excess reagents, secondary antibodies bound to their targets are then detected by chemiluminescence upon incubation with peroxidase reactive substrates. Charge heterogeneity as determined by chemiluminescence was similar to that measured by conventional CIEF technology with absorbance detection for purified mAbs and contaminated mAbs derived directly from host cellular extract. Upon method optimization, the automated CIEF immunoassay was applied to several mAbs of varying isoelectric points, demonstrating the suitability of NanoPro as a rugged high-throughput product characterization tool. Furthermore, qualification of detection sensitivity, precision, and dynamic range are reported with discussion of its advantages as an alternative approach to rapidly characterize charge variants during process development of mAbs.
表征重组单克隆抗体 (mAb) 的电荷异质性需要高通量分析方法，以支持克隆选择和配方筛选。我们在早期工艺开发中应用了 NanoPro 技术来快速测量 mAb 的相对电荷分布。NanoPro 是一种基于多通道毛细管的等电免疫分析，具有整柱成像检测。与传统的毛细管等电聚焦 (CIEF) 平台相比，该检测具有特异性、速度和灵敏度优势。在 CIEF 之后，电荷变异体通过光化学固定在短涂层毛细管的壁上。一旦固定，使用与辣根过氧化物酶偶联的二级抗 IgG 探测 mAb。冲洗掉多余的试剂后，与过氧化物酶反应性底物孵育时，与靶标结合的二级抗体通过化学发光检测。通过化学发光测定的电荷异质性与通过传统 CIEF 技术与吸收检测测定的纯化 mAb 和直接从宿主细胞提取物中衍生的污染 mAb 的电荷异质性相似。在方法优化后，自动化 CIEF 免疫分析应用于几种等电点不同的 mAb，证明了 NanoPro 作为一种坚固的高通量产品特性分析工具的适用性。此外，还报告了检测灵敏度、精密度和动态范围的资格认证，并讨论了其作为在 mAb 工艺开发过程中快速表征电荷变异体的替代方法的优势。
