Assessment of the performance of MAM vs conventional QC methods for evaluation of Product Quality Attributes of adalimumab and etanercept

摘要 单抗和其他生物疗法会受到各种各样的修饰，这些修饰可能会影响 活性和稳定性，因此必须作为质量控制和可比性的一部分进行分析。质谱学 (MS)因其检测蛋白质的能力而成为生物制药分析实验室的主力 在分子水平上的修饰。在过去的几年中，多属性法(MAM)获得了 整个药物开发和QC实验室，几个开发人员实现了一些 MAM在表征或发布中的形式。虽然更换多个QC测试提供了一个机会 简化实验室工作，减少开发时间和审批后成本，仍然存在几个挑战。 虽然一些大型生物制药公司正在QC中实施MAM，但MAM在 生物相似和小型生物制药公司。这项提案涉及考虑的关键领域之一 根据FDA工作人员2019年出版的出版物《MAM的绩效》中概述的在QC中实施MAM 与传统方法进行比较。收集数据以支持从传统技术过渡到MAM是一种 可阻止或延缓生物仿制药开发的重大投资。 这项工作的目标是评估基于MS的MAM与传统QC的性能 识别产品质量属性(PQA)在强制降级时的变化并相互关联的方法 这些PQA在生物活性、结合亲和力和结构上的变化。这项研究的结果将有助于 通过创建可降低障碍的知识库，从传统技术过渡到MAM 采用MAM，并使生物相似制造商能够更广泛地使用MAM。 这里提出的工作将评估和比较单抗(Adalimumab)和Fc的PQAS 融合蛋白(依那西普)从三个不同的来源获得，使用传统的QC方法和 基于MAM的方法。每种技术在强制降级过程中对变化的敏感度将是 评估并与使用基于细胞的分析检测到的功能和结构变化进行比较，结合 分析和圆二色谱。我们建议首先进行阿达利单抗和阿达利单抗的强制降解研究 来自多个来源的依那西普(具体目标1)。PQAS将使用常规方法进行评估，以 评估电荷变体、糖基化和大小变体(特定目标2)，并与特定目标进行比较 使用基于MS的MAM工作流程(特定目标3)检测到的分子修饰以评估可比性 以及对PQAS变化的敏感性。最后，在具体目标4下，我们将评估其生物活性和结构 在PQAS中表现出显著差异的生物治疗产品将分子变化与 结构和功能的变化。

ABSTRACT Monoclonal antibodies and other biotherapeutics are subject to a variety of modifications that can impact activity and stability and therefore must be analyzed as part of QC and comparability. Mass spectrometry (MS) has become a workhorse for biopharmaceutical analytical laboratories due to its ability to detect protein modifications at a molecular level. Over the past few years, the Multi-Attribute Method (MAM) has gained traction throughout pharmaceutical development and QC labs, with several developers implementing some form of MAM in characterization or release. While replacing multiple QC tests provides an opportunity to streamline lab work and decrease development time and post-approval costs, several challenges remain. While some large biopharma companies are implementing MAM in QC, MAM is not as commonly used in biosimilar and small biopharma companies. This proposal addresses one of the key areas of consideration for implementation of MAM in QC as outlined in a 2019 publication from FDA staff: the performance of MAM vs conventional methods. Collecting data to support transitioning from conventional techniques to MAM is a significant investment that can prevent or delay development of biosimilars. The objective of this work is to assess the performance of the MS-based MAM versus conventional QC methods to identify changes in product quality attributes (PQAs) upon forced degradation and to correlate changes in those PQAs with bioactivity, binding affinity, and structure. Results of this study will help support transitioning from conventional techniques to MAM by creating a knowledge base that can lower the barrier to adoption of MAM and enable wider use of MAM by biosimilar manufacturers. The work proposed here will assess and compare PQAs of a monoclonal antibody (adalimumab) and Fc fusion protein (etanercept) acquired from three different sources using both conventional QC methods and MAM-based approaches. Sensitivity of each technique to changes during forced degradation will be evaluated and compared to functional and structural changes detected using cell-based assays, binding assays and circular dichroism. We propose to first conduct forced degradation studies of adalimumab and etanercept from multiple sources (Specific Aim 1). PQAs will be evaluated using conventional methods to assess charge variants, glycosylation, and size variants, (Specific Aim 2) and compared to the specific molecular modifications detected using an MS-based MAM workflow (Specific Aim 3) to assess comparability and sensitivity to changes in PQAs. Lastly, under Specific Aim 4, we will assess the bioactivity and structure of biotherapeutic products exhibiting significant differences in PQAs to correlate molecular changes to changes in structure and function.