Christofi Emilia, O'Hanlon Mark, Curtis Robin, Barman Arghya, Keen Jeff, Nagy Tibor, Barran Perdita
Michael Barber Centre for Collaborative Mass Spectrometry, MBCCMS, Princess Street, Manchester M17DN, U.K.
Manchester Institute of Biotechnology, University of Manchester, Princess Street, Manchester M17DN, U.K.
J Am Soc Mass Spectrom. 2025 Jan 1;36(1):44-57. doi: 10.1021/jasms.4c00253. Epub 2024 Nov 21.
Post expression from the host cells, biotherapeutics undergo downstream processing steps before final formulation. Mass spectrometry and biophysical characterization methods are valuable for examining conformational and stoichiometric changes at these stages, although typically not used in biomanufacturing, where stability is assessed via bulk property studies. Here we apply hybrid MS methods to understand how solution condition changes impact the structural integrity of a biopharmaceutical across the processing pipeline. As an exemplar product, we use the model IgG1 antibody, mAb4. Flexibility, stability, aggregation propensity, and bulk properties are evaluated in relation to perfusion media, purification stages, and formulation solutions. Comparisons with Herceptin, an extensively studied IgG1 antibody, were conducted in a mass spectrometry-compatible solution. Despite presenting similar charge state distributions (CSD) in native MS, mAb4, and Herceptin show distinct unfolding patterns in activated ion mobility mass spectrometry (aIM-MS) and differential scanning fluorimetry (DSF). Herceptin's greater structural stability and aggregation onset temperature () are attributed to heavier glycosylation and kappa-class light chains, unlike the lambda-class light chains in mAb4. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) revealed that mAb4 undergoes substantial structural changes during purification, marked by high flexibility, low melting temperature (Tm), and prevalent repulsive protein-protein interactions but transitions to a compact and stable structure in high-salt and formulated environments. Notably, in formulation, the third constant domain (CH3) of the heavy chain retains flexibility and is a region of interest for aggregation. Future work could translate features of interest from comprehensive studies like this to targeted approaches that could be utilized early in the development stage to aid in decision-making regarding targeted mutations or to guide the design space of bioprocesses and formulation choices.
在从宿主细胞表达后，生物治疗药物在最终制剂之前要经历下游加工步骤。质谱和生物物理表征方法对于检查这些阶段的构象和化学计量变化很有价值，尽管通常不用于生物制造，在生物制造中，稳定性是通过整体性质研究来评估的。在这里，我们应用混合质谱方法来了解溶液条件变化如何影响生物制药在整个加工流程中的结构完整性。作为一个示例产品，我们使用模型IgG1抗体mAb4。针对灌注介质、纯化阶段和制剂溶液，评估了其灵活性、稳定性、聚集倾向和整体性质。在与质谱兼容的溶液中，将其与广泛研究的IgG1抗体赫赛汀进行了比较。尽管在原生质谱中mAb4和赫赛汀呈现出相似的电荷态分布（CSD），但在活化离子淌度质谱（aIM-MS）和差示扫描荧光法（DSF）中，mAb4和赫赛汀显示出不同的解折叠模式。赫赛汀更高的结构稳定性和聚集起始温度（）归因于其更重的糖基化和κ类轻链，这与mAb4中的λ类轻链不同。氢-氘交换质谱（HDX-MS）显示，mAb4在纯化过程中经历了显著的结构变化，其特点是高灵活性、低熔解温度（Tm）和普遍存在的排斥性蛋白质-蛋白质相互作用，但在高盐和制剂环境中转变为紧凑且稳定的结构。值得注意的是，在制剂中，重链的第三个恒定结构域（CH3）保持灵活性，是聚集的一个关注区域。未来的工作可以将此类综合研究中感兴趣的特征转化为有针对性的方法，这些方法可在开发阶段早期使用，以协助做出关于靶向突变的决策，或指导生物工艺和制剂选择的设计空间。
