Formulation Development, Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10591, USA.
Anal Bioanal Chem. 2024 Dec;416(29):7173-7182. doi: 10.1007/s00216-024-05623-z. Epub 2024 Nov 1.
Adeno-associated virus (AAV) vectors are widely used to deliver therapeutic transgenes due to their superior safety, relatively low immunogenicity, and ability to target diverse tissues. AAV gene therapy products are typically formulated as frozen liquid and stored below - 60 °C, and therefore are subjected to multiple freeze/thaw cycles during manufacturing and administration. Recent studies have shown that genome DNA leakage could be induced by freeze/thaw stress. DNA leakage from AAV capsids has been reported to potentially impact product stability, induce immune responses, and compromise product efficacy. Thus, further characterization to improve the understanding of genome DNA leakage is necessary for mitigating the risks associated with genome DNA leakage during AAV product development. In this work, we developed an optimized size-exclusion chromatography (SEC) method for quantifying the leakage of genome DNA across multiple different AAV serotypes and demonstrated satisfactory assay performance in sensitivity, precision, and linearity. Furthermore, we showed that this method could also be applied to quantifying additional quality attributes of AAV, including the percentage of full capsids and quantification of AAV dimers. By using this optimized SEC method, we demonstrated that significantly increased free DNA was observed with increasing freeze/thaw cycles or at a temperature approaching the onset temperature for genome DNA ejection, which was effectively mitigated by the addition of 1.5% w/v sucrose in the AAV formulation. Thus, this optimized SEC method can serve as an invaluable tool for AAV formulation, product, and process development in ensuring the quality and stability of AAV gene therapy products.
腺相关病毒 (AAV) 载体由于其优越的安全性、相对较低的免疫原性和靶向多种组织的能力，被广泛用于递送治疗性转基因。AAV 基因治疗产品通常被制成冷冻液体，并储存在低于-60°C 的温度下，因此在制造和给药过程中会经历多次冻融循环。最近的研究表明，冻融应激可诱导基因组 DNA 泄漏。已经有报道称，AAV 衣壳内的 DNA 泄漏可能会影响产品稳定性、引发免疫反应并降低产品功效。因此，进一步的特征分析对于减轻 AAV 产品开发过程中与基因组 DNA 泄漏相关的风险是必要的。在这项工作中，我们开发了一种优化的尺寸排阻色谱 (SEC) 方法，用于定量测定多种不同 AAV 血清型的基因组 DNA 泄漏情况，并证明了该方法在灵敏度、精密度和线性方面具有令人满意的检测性能。此外，我们还表明，该方法还可用于定量测定 AAV 的其他质量属性，包括完整衣壳的百分比和 AAV 二聚体的定量。通过使用这种优化的 SEC 方法，我们证明，随着冻融循环的增加或接近基因组 DNA 弹出的起始温度，游离 DNA 的量显著增加，而在 AAV 制剂中添加 1.5%w/v 蔗糖可有效减轻这种情况。因此，这种优化的 SEC 方法可以作为 AAV 制剂、产品和工艺开发的宝贵工具，以确保 AAV 基因治疗产品的质量和稳定性。
