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

项目摘要 合成（短）寡核苷酸是治疗和预防各种各样的肿瘤的令人兴奋的一类疗法。 疾病，包括传统药物开发努力失败的疾病。与天然核酸相比， 酸，其是酶促制备的，合成的寡核苷酸药物是通过固相化学制备的， 化学合成固相化学合成为引入化学物质提供了有利的平台， 在药物开发过程中进行修饰以克服天然核酸的不良药理学性质。 寡核苷酸药物的常见化学修饰涉及天然寡核苷酸中的一个氧的替换。 磷酸二酯（PO）键与硫形成硫代磷酸酯（PS）键。PS链接增强 通过增加核酸酶抗性和促进蛋白质结合而具有药理学性质。PS修改 通常用于反义寡核苷酸（ASO）治疗，反义寡核苷酸是药物发现的普遍平台 其中几个ASO获得了美国食品和药物管理局（FDA）的批准。的后果 用PS键化学取代PO键是在磷原子上产生手性中心。 这导致PS中心具有两种立体化学构型。潜在PS非对映异构体的数量 是2n，其中n是寡核苷酸中PS键的数目。对于具有19个PS的20-mer寡核苷酸， 这相当于524，288种潜在的立体异构体。尽管ASO药物越来越受欢迎， 迫切需要开发高分辨率的分析方法， 在生产和仿制药中用于质量控制目的的寡核苷酸中的非对映体组合物 发展因此，本提案的目的是开发新的创新分析方法 用于分析ASO药物中的非对映体组成。我们将使用TEGSEDI作为模型ASO制剂。 我们的建议将确定生产过程是否以及如何影响非对映异构体 制剂中的分布。我们打算通过制定一个 多维的分析方法，计算集成了最先进的液相色谱法， 质谱（MS）和核磁共振（NMR）光谱。我们的实验方法将 包括inotersen（TEGSEDI通用名）和短链片段的内部化学合成， 研究化学合成的效果（即，制造）对PS连接中的立体化学结果有影响 阵我们将使用数据科学，包括多变量分析和聚类，以评估非对映异构体 组合物，并应用机器学习技术来预测TEGSEDI和TEGSEDI的非对映体组合物。 我们的内部准备inotersen。我们开发的方法将直接转移到其他合成 这将极大地促进仿制寡核苷酸药物的开发和评估。 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.