Amgen Research, Amgen, Inc. 360 Binney Street, Cambridge, MA, 02141, USA.
Amgen Research, Amgen, Inc. One Amgen Center Drive, Thousand Oaks, CA, 91320, USA.
J Chromatogr A. 2022 Jan 4;1661:462679. doi: 10.1016/j.chroma.2021.462679. Epub 2021 Nov 20.
N-acetylgalactosamine (GalNAc)-modified small interfering ribonucleic acids (siRNA) have shown promising outcomes for targeted siRNA delivery resulting in gene silencing in vivo; however, their structural complexity requires development of new purification methods to address high purity and recovery requirements. The current study evaluates complementary purification approaches using a mixed-mode Scherzo SS-C18 and anion-exchange (AEX) TSK-gel SuperQ-5PW for a range of single-stranded triantennary GalNAc-oligonucleotides. Initially, the semi-preparative mixed-mode support (10 × 250 mm, 3 µm) was compared against the preparative AEX analogue (21.5 × 300 mm, 13 µm), with the former affording double the recovery and higher purity of 95% over its AEX counterpart displaying 91% for a selected siRNA conjugate. An assortment of GalNAc-modified oligonucleotides was later purified using the mixed-mode resin revealing good recoveries (∼30-60%) and high purities of 90-94% ranging from straightforward to more challenging purifications. High sample loading in the 20 mg range was achieved, which was comparable with the larger preparative TSKgel SuperQ-5PW support. The Scherzo-SS-C18 resin also afforded some degree of resolution between diastereomers containing phosphorothioate functionalities. The TSKgel SuperQ-5PW support was later investigated to provide orthogonal separation selectivity to the Scherzo-SS-C18 column enabling purification of a selected, GalNAc-siRNA conjugate. The developed pH (8.5-11) and salt (0.3-0.7 M) gradients method provided enhanced separation selectivity between the free and conjugated siRNA, while minimizing formation of secondary structures and highlighting a complementary approach to deal with challenging purifications of oligonucleotide-GalNAc conjugates. Together, the use of AEX and mixed-mode columns provide much needed orthogonality to deal with complex GalNAc-modified oligonucleotides and potentially other upcoming modalities.
N-乙酰氨基半乳糖（GalNAc）修饰的小干扰核糖核酸（siRNA）在体内靶向 siRNA 递送上显示出有前景的结果，导致基因沉默；然而，它们的结构复杂性需要开发新的纯化方法来满足高纯度和回收率的要求。本研究评估了使用混合模式 Scherzo SS-C18 和阴离子交换（AEX）TSK-gel SuperQ-5PW 对一系列单链三触角 GalNAc 寡核苷酸的互补纯化方法。最初，半制备混合模式载体（10×250mm，3μm）与制备型 AEX 类似物（21.5×300mm，13μm）进行比较，前者的回收率是后者的两倍，纯度高达 95%，而 AEX 类似物的纯度为 91%，对于选定的 siRNA 缀合物。随后，使用混合模式树脂对各种 GalNAc 修饰的寡核苷酸进行了纯化，结果显示回收率良好（约 30-60%），纯度高达 90-94%，范围从简单到更具挑战性的纯化。实现了 20mg 范围内的高样品负载，与更大的制备型 TSKgel SuperQ-5PW 载体相当。Scherzo-SS-C18 树脂还在一定程度上实现了含有硫代磷酸酯官能团的非对映异构体之间的分辨率。随后研究了 TSKgel SuperQ-5PW 载体，以提供与 Scherzo-SS-C18 柱的正交分离选择性，从而实现选定的 GalNAc-siRNA 缀合物的纯化。开发的 pH（8.5-11）和盐（0.3-0.7M）梯度方法提供了自由和共轭 siRNA 之间增强的分离选择性，同时最大限度地减少了二级结构的形成，并突出了一种互补方法来处理寡核苷酸-GalNAc 缀合物的挑战性纯化。总之，使用 AEX 和混合模式柱提供了急需的正交性，可以处理复杂的 GalNAc 修饰的寡核苷酸和潜在的其他新兴模式。
