Bramham Jack E, Podmore Adrian, Davies Stephanie A, Golovanov Alexander P
Manchester Institute of Biotechnology and School of Chemistry, Faculty of Science and Engineering, The University of Manchester, Manchester M1 7DN, U.K.
Dosage Form Design & Development, BioPharmaceuticals Development, R&D, AstraZeneca, Cambridge CB21 6GH, U.K.
ACS Pharmacol Transl Sci. 2020 Dec 16;4(1):288-295. doi: 10.1021/acsptsci.0c00188. eCollection 2021 Feb 12.
Biopharmaceutical proteins are important drug therapies in the treatment of a range of diseases. Proteins, such as antibodies (Abs) and peptides, are prone to chemical and physical degradation, particularly at the high concentrations currently sought for subcutaneous injections, and so formulation conditions, including buffers and excipients, must be optimized to minimize such instabilities. Therefore, both the protein and small molecule content of biopharmaceutical formulations and their stability are critical to a treatment's success. However, assessing all aspects of protein and small molecule stability currently requires a large number of analytical techniques, most of which involve sample dilution or other manipulations which may themselves distort sample behavior. Here, we demonstrate the application of H nuclear magnetic resonance (NMR) spectroscopy to study both protein and small molecule content and stability in high-concentration (100 mg/mL) Ab formulations. We show that protein degradation (aggregation or fragmentation) can be detected as changes in 1D H NMR signal intensity, while apparent relaxation rates are specifically sensitive to Ab fragmentation. Simultaneously, relaxation-filtered spectra reveal the presence and degradation of small molecule components such as excipients, as well as changes in general solution properties, such as pH. H NMR spectroscopy can thus provide a holistic overview of biopharmaceutical formulation content and stability, providing a preliminary characterization of degradation and acting as a triaging step to guide further analytical techniques.
生物制药蛋白质是治疗一系列疾病的重要药物疗法。蛋白质，如抗体（Abs）和肽，容易发生化学和物理降解，特别是在目前皮下注射所追求的高浓度下，因此必须优化包括缓冲液和辅料在内的制剂条件，以尽量减少此类不稳定性。因此，生物制药制剂中的蛋白质和小分子含量及其稳定性对于治疗的成功至关重要。然而，目前评估蛋白质和小分子稳定性的所有方面需要大量分析技术，其中大多数涉及样品稀释或其他可能本身会扭曲样品行为的操作。在这里，我们展示了氢核磁共振（NMR）光谱在研究高浓度（100 mg/mL）抗体制剂中的蛋白质和小分子含量及稳定性方面的应用。我们表明，蛋白质降解（聚集或碎片化）可以通过一维氢核磁共振信号强度的变化来检测，而表观弛豫率对抗体碎片化特别敏感。同时，弛豫滤波光谱揭示了辅料等小分子成分的存在和降解，以及诸如pH值等一般溶液性质的变化。因此，氢核磁共振光谱可以提供生物制药制剂含量和稳定性的全面概述，提供降解的初步表征，并作为一个分流步骤来指导进一步的分析技术。
