Rodenstein Carina, Schmid Eva, Markova Natalia, Seidl Andreas
Leukocare AG, Martinsried, Germany.
Malvern Panalytical Ltd., Malvern, Worcestershire, United Kingdom.
Microbiol Spectr. 2025 Jun 30:e0180624. doi: 10.1128/spectrum.01806-24.
Formulation development for gene therapy viral vectors requires high-performance analytical methods that indicate or predict virus stability. Additionally, if testing involves multiple excipients, sample throughput must be sufficiently high, and the method should require low amounts of virus material. These are challenging and sometimes conflicting requirements, especially in the analysis of adeno-associated viral vectors, which are currently the most prominent viral vectors in gene therapy development. In this exploratory study, we evaluated the ability of different analytical methods to indicate or predict the stability of the adeno-associated viral vectors and identified highly suitable techniques that fulfilled the requirements for analysis during formulation development. As functionality assays, such as reporter gene assays, are already established for rapid quality control assessments, they are not addressed in this study. From this study, we conclude that two methods have great potential to predict the stability of adeno-associated viral vectors: extrinsic differential scanning fluorimetry using SYBR Gold as a DNA binding fluorescent dye to detect genome release and nano differential scanning fluorimetry to investigate capsid unfolding behavior (based on intrinsic protein fluorescence). Size-exclusion chromatography, using multi-angle light scattering and UV detection, was found to be stability-indicating for particle size distribution and determining genome load of adeno-associated viral vectors.IMPORTANCEViral vectors are of growing importance in the areas of gene therapy, oncology, and vaccine development. However, these vectors are very unstable and usually have to be stored frozen at very low temperatures due to sub-optimal formulation conditions, in many cases. The development of superior formulations for viral vectors requires high-performance analytical methods. In this study, we evaluated relevant analytical methods with respect to sample throughput, material consumption, and applicability for viral vector formulation development. To our knowledge, this is the first time that methods for viral vector analysis were categorized according to their power to predict or indicate time-dependent long-term stability. This categorization of analytical methods is essential to rationalize, accelerate, and enhance the formulation development of viral vectors. Therefore, the studies in this article are prerequisites for the development of more stable viral vectors for gene therapy and vaccines and higher yields in manufacturing.
基因治疗病毒载体的制剂开发需要高性能的分析方法来指示或预测病毒稳定性。此外，如果测试涉及多种辅料，样品通量必须足够高，并且该方法应需要少量的病毒材料。这些要求具有挑战性，有时甚至相互冲突，特别是在分析腺相关病毒载体时，腺相关病毒载体目前是基因治疗开发中最突出的病毒载体。在这项探索性研究中，我们评估了不同分析方法指示或预测腺相关病毒载体稳定性的能力，并确定了在制剂开发过程中满足分析要求的高度适用技术。由于报告基因检测等功能检测方法已用于快速质量控制评估，因此本研究未涉及。从这项研究中，我们得出结论，有两种方法具有预测腺相关病毒载体稳定性的巨大潜力：使用SYBR Gold作为DNA结合荧光染料来检测基因组释放的外在差示扫描荧光法，以及用于研究衣壳解折叠行为的纳米差示扫描荧光法（基于内在蛋白质荧光）。发现使用多角度光散射和紫外检测的尺寸排阻色谱法可指示腺相关病毒载体的粒径分布并确定基因组负载量。
重要性：病毒载体在基因治疗、肿瘤学和疫苗开发领域的重要性日益增加。然而，这些载体非常不稳定，在许多情况下，由于制剂条件不理想，通常必须在极低温度下冷冻保存。开发用于病毒载体的优质制剂需要高性能的分析方法。在本研究中，我们从样品通量、材料消耗和对病毒载体制剂开发的适用性方面评估了相关分析方法。据我们所知，这是首次根据分析方法预测或指示时间依赖性长期稳定性的能力对病毒载体分析方法进行分类。这种分析方法的分类对于使病毒载体制剂开发合理化、加速和加强至关重要。因此，本文中的研究是开发用于基因治疗和疫苗的更稳定病毒载体以及提高生产产量的先决条件。
