Sodium Dodecyl Sulfate Removal Interface to Enable Characterization of Fragment Impurities in Monoclonal Antibodies by Capillary Electrophoresis Sodium Dodecyl Sulfate Coupled to Mass Spectrometry

计划摘要 单抗(MAbbs)已成为人类免疫缺陷的主流治疗蛋白。 生物制药行业(生物制药)，以及对高效分析工具的需求 表征和质量控制继续增加。在制造和储存过程中，裂解 单抗的一级结构可能是由于不同的碎裂诱导机制而产生的，而由此产生的 碎片种类可能会对产品的安全性和有效性产生负面影响。因此， 碎裂是常规监测以评估单抗纯度和完整性的关键质量属性 以及单抗衍生的生物制品从生产到商业化。 以十二烷基硫酸钠为毛细管的毛细管电泳法 模拟聚丙烯酰胺凝胶电泳法(SDS-PAGE)被广泛用于mAb的监测 在生产、配方、稳定性和商业释放过程中的碎片。CE-SDS使用SDS来 变性蛋白质，并使非共价结合的亚基可分离。分离的基础是 由十二烷基硫酸钠结合引起的表面电荷驱动的电泳迁移，它与 蛋白质的相对分子质量。在筛选凝胶基质中，实现了基于尺寸的分离，因为所有的十二烷基硫酸酯-十二烷基硫酸酯- 蛋白质复合体具有相似的电荷尺寸比。CE-十二烷基硫酸钠提供了良好的碎片分辨率， 但缺乏通过直接耦合到已分解碎片的质谱学来进行质量鉴定 目前在生物制药领域造成了重大缺口。 GMJ Technologies(GMJ)正在申请SBIR第一阶段资金，以开发微型SDS 耗竭电泳(SDE)接口，允许CE-十二烷基硫酸酯直接连接到质谱仪(MS)。 建议的接口将使用自由流区带电泳机制来在线去除 电喷雾电离质谱仪(ESI-MS)前的CE-十二烷基硫酸钠(CE-十二烷基硫酸钠)峰。有了这项创新， GMJ的目标是解决单抗从研究到质量控制方面的一个关键需求 商业化。通过省去几个费力的步骤，所提出的装置将显著改进 提高了单抗片段表征工作流程的效率，提高了分析精度， 敏感性，并为生物制药研究人员提供节省成本的好处。

PROGRAM SUMMARY Monoclonal antibodies (mAbs) have become mainstream therapeutic proteins in the biopharmaceutical industry (biopharma), and the demand for efficient analytical tools for their characterization and quality control continues to increase. During manufacturing and storage, cleavage of mAb’s primary structure can occur due to different fragmentation induced mechanisms, and the resulting fragment species can have negative implication for the safety and efficacy of the product. Therefore, fragmentation is a critical quality attribute routinely monitored to assess the purity and integrity of mAbs and mAb-derived biologics from production to commercialization. Capillary electrophoresis with sodium dodecyl sulfate (CE-SDS), which is the capillary analogue of polyacrylamide gel electrophoresis (SDS-PAGE), is widely employed for monitoring mAb fragments during production, formulation, stability, and commercial release. CE-SDS employs SDS to denature proteins and render non-covalently associated subunits separable. The separation is based on electrophoretic migration driven by the surface charge induced by SDS binding, which is proportional to the protein’s molecular weight. In a sieving gel matrix, a size-based separation is achieved since all SDS- protein complexes have similar charge-to-size ratio. CE-SDS provides excellent resolution of fragments, but lack of mass identification through direct coupling to mass spectrometry of the resolved fragments currently creates a major gap in the biopharma. GMJ Technologies (GMJ) is requesting SBIR Phase I funding to develop a micro SDS depletion electrophoresis (SDE) interface to allow direct coupling of CE-SDS to mass spectrometry (MS). The proposed interface will use free-flow zone electrophoresis mechanism to remove SDS in-line from the CE-SDS peaks of interest prior to electrospray ionization mass spectrometry (ESI-MS). With this innovation, GMJ aims to address a critical need in the characterization and quality control of mAbs from research to commercialization. By eliminating several laborious steps, the proposed device will significantly improve the efficiency in the characterization workflow for mAbs’ fragments, improve the analysis precision and sensitivity, and provide cost-saving benefits for biopharma researchers.