Process Research and Development, Merck & Co., Inc., Rahway, NJ, United States.
Cytiva, Björkgatan 30, Uppsala, Sweden.
J Chromatogr A. 2024 Feb 22;1717:464670. doi: 10.1016/j.chroma.2024.464670. Epub 2024 Feb 1.
Increased demand for mRNA-based therapeutics and improved in vitro transcription (IVT) yields have challenged the mRNA purification platform. Hybridization-affinity chromatography with an immobilized oligo-deoxythymidilic acid (oligodT) ligand is often used to capture mRNA through base pairing with the polyadenylated tail. Commercially available oligodT matrices include perfusive cross-linked poly(styrene-divinylbenzene) 50 µm POROS™ chromatography resin beads and convective polymethacrylate CIMmultus® monolithic columns consisting of 2 µm interconnected channels. POROS™ columns may be limited by poor mass transfer for larger mRNAs and slow flowrates, while monoliths can operate at higher flowrates but are limited by modest binding capacity. To enable both high flowrates and binding capacity for mRNA of all lengths, prototype chromatography media was developed by Cytiva using oligodT immobilized electrospun cellulose nanofibers (Fibro™) with a 0.3-0.4 µm pore size. In this work, four polyadenylated mRNAs ranging from ∼1900-4300 nucleotides were used to compare the dynamic binding capacity (DBC) of Fibro™, POROS® and CIMmultus® columns as a function of residence time and binding buffer compositions. Fibro™ improved the DBC ∼2-4-fold higher than CIMmultus® and ∼2-13-fold higher than POROS™ across all residence times, mRNA length, and binding matrix compositions tested. CIMmultus® DBC was least dependent on residence time and mRNA size, while both Fibro™ and POROS™ DBC increased at slower flowrates and with shorter mRNA. Surprisingly, inverse size exclusion (ISE) experiments showed that POROS™ was not limited by diffusion and POROS™ along with CIMmultus® demonstrate higher mRNA permeation however the Fibro™ prototype is not in the final configuration. Lastly, IVT reaction products were subjected to purification and oligodT elution product yield, quality, and purity were consistent across the three matrices investigated. These results highlight the benefits of high DBC and equivalent product profiles offered by the oligodT Fibro™ prototype compared to commercially available oligodT media.
对基于 mRNA 的治疗药物的需求增加和体外转录 (IVT) 产量的提高给 mRNA 纯化平台带来了挑战。通过与聚腺苷酸化尾巴的碱基配对，固定化寡脱氧胸苷 (oligodT) 配体的杂交亲和层析通常用于捕获 mRNA。商业上可用的寡 dT 基质包括可渗透交联的聚苯乙烯-二乙烯基苯 50 µm POROS™ 色谱树脂珠和由 2 µm 相互连通的通道组成的对流聚甲基丙烯酸酯 CIMmultus®整体柱。POROS™ 柱可能受到较大 mRNA 的传质不良和较慢流速的限制，而整体柱可以在较高流速下运行，但受到适度结合能力的限制。为了实现所有长度的 mRNA 的高通量和结合能力，Cytiva 使用固定化寡脱氧胸苷的静电纺丝纤维素纳米纤维 (Fibro™) 开发了原型色谱介质，其孔径为 0.3-0.4 µm。在这项工作中，使用了四种长度从约 1900-4300 个核苷酸的聚腺苷酸化 mRNA，以比较 Fibro™、POROS®和 CIMmultus® 柱作为停留时间和结合缓冲液组成函数的动态结合容量 (DBC)。Fibro™ 在所有停留时间、mRNA 长度和结合基质组成下，将 DBC 提高了 2-4 倍，比 CIMmultus®高 2-13 倍，比 POROS™高。CIMmultus®的 DBC 最不依赖于停留时间和 mRNA 大小，而 Fibro™和 POROS™的 DBC 在较慢的流速下和较短的 mRNA 时增加。令人惊讶的是，反尺寸排阻 (ISE) 实验表明 POROS™不受扩散限制，POROS™和 CIMmultus®显示出更高的 mRNA 渗透性，然而 Fibro™原型尚未最终配置。最后，将 IVT 反应产物进行纯化，寡脱氧胸苷洗脱产物的产率、质量和纯度在三种研究基质中是一致的。这些结果突出了与商业上可用的寡 dT 介质相比，高 DBC 和等效产物谱的寡 dT Fibro™ 原型的优势。
