Department of Biochemical Engineering, University College London, London WC1E 6BT, United Kingdom.
LGC, Queens Road, Teddington, Middlesex TQ11 0LY, United Kingdom.
Mol Pharm. 2024 Apr 1;21(4):1965-1976. doi: 10.1021/acs.molpharmaceut.3c01211. Epub 2024 Mar 22.
Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating factor (G-CSF) formulations, confirming predictions made using computational structure docking. More recently, solid-state HDX mass spectrometry (ssHDX-MS) was developed for proteins in the lyophilized state. Deuterium uptake in ssHDX-MS has been shown for various proteins, including monoclonal antibodies, to be highly correlated with storage stability, as measured by protein aggregation and chemical degradation. As G-CSF is known to lose activity through aggregation upon lyophilization, we applied the ssHDX-MS method with peptide mapping to four different lyophilized formulations of G-CSF to compare the impact of three excipients on local structure and exchange dynamics. HDX at 22 °C was confirmed to correlate well with the monomer content remaining after lyophilization and storage at -20 °C, with sucrose providing the greatest protection, and then phenylalanine, mannitol, and no excipient leading to progressively less protection. Storage at 45 °C led to little difference in final monomer content among the formulations, and so there was no discernible relationship with total deuterium uptake on ssHDX. Incubation at 45 °C may have led to a structural conformation and/or aggregation mechanism no longer probed by HDX at 22 °C. Such a conformational change was observed previously at 37 °C for liquid-formulated G-CSF using NMR. Peptide mapping revealed that tolerance to lyophilization and -20 °C storage was linked to increased stability in the small helix, loop AB, helix C, and loop CD. LC-MS HDX and NMR had previously linked loop AB and loop CD to the formation of a native-like state (N*) prior to aggregation in liquid formulations, suggesting a similar structural basis for G-CSF aggregation in the liquid and solid states.
氢/氘交换质谱（HDX-MS）先前阐明了赋形剂与液体粒细胞集落刺激因子（G-CSF）制剂中蛋白质之间的相互作用，证实了使用计算结构对接做出的预测。最近，发展了用于冷冻干燥状态下蛋白质的固态 HDX 质谱（ssHDX-MS）。已证明 ssHDX-MS 中各种蛋白质（包括单克隆抗体）的氘摄入与储存稳定性高度相关，如通过蛋白质聚集和化学降解来测量。由于 G-CSF 在冷冻干燥时通过聚集而失去活性，因此我们应用 ssHDX-MS 方法结合肽图分析了四种不同的 G-CSF 冷冻干燥制剂，以比较三种赋形剂对局部结构和交换动力学的影响。在 22°C 下的 HDX 与冷冻干燥后和在-20°C 下储存时剩余的单体含量很好地相关，其中蔗糖提供最大的保护，其次是苯丙氨酸、甘露醇和无赋形剂，导致保护作用逐渐减弱。在 45°C 下储存对制剂最终单体含量几乎没有差异，因此与 ssHDX 上的总氘摄入没有明显的关系。在 45°C 下孵育可能导致结构构象和/或聚集机制不再通过 22°C 下的 HDX 探测。以前在 37°C 下使用 NMR 观察到对于液体配方的 G-CSF 发生了这种构象变化。肽图分析表明，对冷冻干燥和-20°C 储存的耐受性与小螺旋、AB 环、C 螺旋和 CD 环中的稳定性增加有关。LC-MS HDX 和 NMR 先前将 AB 环和 CD 环与在液体配方中聚集之前形成类似天然状态（N*）联系起来，这表明在液体和固体状态下 G-CSF 聚集具有类似的结构基础。
