Multidimensional analytical and computational approach to determine diastereomer compositions in oligonucleotide drug products

项目摘要 合成(短)寡核苷酸是一类令人兴奋的治疗方法，用于治疗和预防各种 疾病，包括传统药物开发努力失败的疾病。与天然核不同 酸是通过酶法制备的，合成的寡核苷酸药物是通过固相化学方法制备的 化学合成。固相化学合成为引进化学物质提供了有利平台 在药物开发过程中进行修改，以克服天然核酸较差的药理特性。 寡核苷酸药物的一种常见的化学修饰包括取代天然的一个氧 磷酸二酯(PO)与硫磺形成硫代硫酸酯(PS)键。PS链接增强 通过增加核酸酶抗性和促进蛋白质结合的药理特性。PS的改性 通常用于反义寡核苷酸(ASO)疗法，这是药物发现的普遍平台 有几个ASO获得了美国食品和药物管理局(FDA)的批准。其后果是 用PS键化学取代PO键是在磷原子上创建手性中心。 这导致PS中心具有两种立体化学构型。潜在的PS非对映异构体的数量 为2n，其中n为寡核苷酸中PS键的数目。对于具有19ps的20聚体寡核苷酸 链接这相当于524,288个潜在的立体异构体。尽管ASO药物越来越受欢迎，但有 迫切需要开发高分辨率的分析方法来全面表征 生产和仿制药质量控制用寡核苷酸中的非对映异构体组合物 发展。因此，这项提议的目的是开发新的和创新的分析方法 用于分析ASO药物中的非对映异构体成分。我们将使用TegSEDI作为我们的ASO药物产品模型。 我们的建议将决定制造过程是否以及如何影响非对映异构体 在药品中的分布。我们打算通过制定一项 多维分析方法，通过计算集成了最先进的液相色谱仪， 质谱仪(MS)和核磁共振(核磁共振)光谱分析。我们的实验方法将 包括伊诺特森(TEGSEDI通称)和短链片段的内部化学合成以 研究化学合成(即制造)对PS连接中立体化学结果的影响 队形。我们将使用包括多元分析和聚类在内的数据科学来评估非对映异构体 并应用机器学习技术预测TEGSEDI和TEGSEDI的非对映异构体组成 我们的内部准备了因特森。我们开发的方法将直接适用于其他合成方法 并将极大地促进非专利寡核苷酸药物的开发和评价 产品。

PROJECT ABSTRACT Synthetic (short) oligonucleotides are an exciting class of therapeutics to treat and prevent a wide variety of diseases, including diseases where traditional drug development efforts have failed. In contrast to native nucleic acids, which are prepared enzymatically, synthetic oligonucleotide drugs are prepared chemically via solid-phase chemical synthesis. Solid-phase chemical synthesis provides an advantageous platform for introducing chemical modifications during drug development to overcome the poor pharmacological properties of native nucleic acids. A common chemical modification for oligonucleotide drugs involves the replacement of one oxygen in the natural phosphodiester (PO) linkage with a sulfur creating a phosphorothioate (PS) linkage. The PS linkage enhances pharmacology properties by increasing nuclease resistance and facilitating protein binding. The PS modification is routinely used in antisense oligonucleotide (ASO) therapeutics that are a prevalent platform for drug discovery with several ASOs gaining United States Food and Drug Administration (FDA) approval. The consequence of chemically replacing the PO linkage with a PS linkage is the creation of a chiral center at the phosphorous atom. This leads to the PS center having two stereochemical configurations. The number of potential PS diastereomers is 2n where n is the number of PS linkages in the oligonucleotide. For a 20-mer oligonucleotide which has 19 PS linkages this equates to 524,288 potential stereoisomers. Despite the increasing popularity of ASO drugs, there is a pressing need to develop high-resolution analytical methods that can comprehensively characterize the diastereomer composition in oligonucleotides for quality control purposes in manufacturing and generic drug development. Consequently, the purpose of this proposal is to develop new and innovative analytical methods for analyzing the diastereomer composition in ASO drugs. We will use TEGSEDI as our model ASO drug product. Our proposal will determine whether and how the manufacturing process may affect the diastereomeric distributions in the drug product. We intend to successfully accomplish these objectives by developing a multidimensional analytical approach that computationally integrates state-of-the-art liquid chromatography, mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Our experimental approach will include in-house chemical synthesis of inotersen (generic name for TEGSEDI) and short chain fragments to investigate the effect chemical synthesis (i.e., manufacturing) has on the stereochemical outcome in PS linkage formation. We will use data science including multivariate analysis and clustering to assess the diastereomer composition and apply machine learning techniques to predict the diastereomer compositions of TEGSEDI and our in-house prepared inotersen. Our developed methods will be directly transferable to other synthetic oligonucleotides and will greatly facilitate the development and assessment of generic oligonucleotide drug products.