Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.
Analytical Development and Attribute Sciences, Biologics Development, Global Product Development and Supply, Bristol Myers Squibb, New Brunswick, New Jersey 08903, United States.
Anal Chem. 2024 Jun 18;96(24):10003-10012. doi: 10.1021/acs.analchem.4c01408. Epub 2024 Jun 9.
Fc-fusion proteins are an emerging class of protein therapeutics that combine the properties of biological ligands with the unique properties of the fragment crystallizable (Fc) domain of an immunoglobulin G (IgG). Due to their diverse higher-order structures (HOSs), Fc-fusion proteins remain challenging characterization targets within biopharmaceutical pipelines. While high-resolution biophysical tools are available for HOS characterization, they frequently demand extended time frames and substantial quantities of purified samples, rendering them impractical for swiftly screening candidate molecules. Herein, we describe the development of ion mobility-mass spectrometry (IM-MS) and collision-induced unfolding (CIU) workflows that aim to fill this technology gap, where we focus on probing the HOS of a model Fc-Interleukin-10 (Fc-IL-10) fusion protein engineered using flexible glycine-serine linkers. We evaluate the ability of these techniques to probe the flexibility of Fc-IL-10 in the absence of bulk solvent relative to other proteins of similar size, as well as localize structural changes of low charge state Fc-IL-10 ions to specific Fc and IL-10 unfolding events during CIU. We subsequently apply these tools to probe the local effects of glycine-serine linkers on the HOS and stability of IL-10 homodimer, which is the biologically active form of IL-10. Our data reveals that Fc-IL-10 produces significantly more structural transitions during CIU and broader IM profiles when compared to a wide range of model proteins, indicative of its exceptional structural dynamism. Furthermore, we use a combination of enzymatic approaches to annotate these intricate CIU data and localize specific transitions to the unfolding of domains within Fc-IL-10. Finally, we detect a strong positive, quadratic relationship between average linker mass and fusion protein stability, suggesting a cooperative influence between glycine-serine linkers and overall fusion protein stability. This is the first reported study on the use of IM-MS and CIU to characterize HOS of Fc-fusion proteins, illustrating the practical applicability of this approach.
Fc 融合蛋白是一类新兴的蛋白治疗药物，它将生物配体的特性与免疫球蛋白 G（IgG）的片段可结晶（Fc）结构域的独特性质结合在一起。由于其多样化的高级结构（HOS），Fc 融合蛋白仍然是生物制药管道中具有挑战性的表征目标。虽然有用于 HOS 表征的高分辨率生物物理工具，但它们通常需要较长的时间框架和大量的纯化样品，因此对于快速筛选候选分子来说不切实际。在此，我们描述了开发离子淌度-质谱（IM-MS）和碰撞诱导解折叠（CIU）工作流程的情况，旨在填补这一技术空白，我们专注于探测使用灵活的甘氨酸-丝氨酸接头工程化的模型 Fc-白细胞介素 10（Fc-IL-10）融合蛋白的 HOS。我们评估了这些技术探测 Fc-IL-10 在没有大量溶剂的情况下相对于其他类似大小的蛋白质的灵活性的能力，以及将低电荷状态 Fc-IL-10 离子的结构变化定位到特定的 Fc 和 IL-10 解折叠事件在 CIU 期间。随后，我们将这些工具应用于探测甘氨酸-丝氨酸接头对 IL-10 同源二聚体 HOS 和稳定性的局部影响，IL-10 同源二聚体是 IL-10 的生物活性形式。我们的数据表明，与广泛的模型蛋白相比，Fc-IL-10 在 CIU 过程中产生了更多的结构转变，并且 IM 谱更宽，表明其具有异常的结构动态性。此外，我们使用组合酶方法对这些复杂的 CIU 数据进行注释，并将特定的转变定位到 Fc-IL-10 内的结构域解折叠。最后，我们检测到平均接头质量与融合蛋白稳定性之间存在强烈的正二次关系，表明甘氨酸-丝氨酸接头与融合蛋白整体稳定性之间存在协同影响。这是首次使用 IM-MS 和 CIU 来表征 Fc 融合蛋白的 HOS 的报道研究，说明了该方法的实际适用性。
