Department of Chemical and Biomolecular Engineering, North Carolina State University, 1017 Main Campus Drive, Centennial Campus, Partner's Building I, Suite 3200, Box 7006, Raleigh, NC 27695-7565, USA.
J Chromatogr A. 2012 Jul 6;1245:55-64. doi: 10.1016/j.chroma.2012.04.072. Epub 2012 May 4.
A strategy of modification of resin surface chemistry is presented to produce hydrophilic peptide-based alkaline-stable affinity adsorbents for the purification of biopharmaceuticals from complex media. In this work, the peptide-based affinity adsorbent HWRGWV-Toyopearl resin for the purification of IgG is presented as an example. When prepared by direct peptide synthesis on the chromatographic matrix, the peptide-based resin showed lability under alkaline conditions. In fact, the regeneration with aqueous 0.1 M NaOH caused the leaching of 40% of the peptide ligand, resulting in a decrease of IgG yield from 85% to 23%. It was found that the ligand leaching was caused by the coupling of a significant amount of peptide by alkaline-labile ester bonds. A method was designed to prevent the formation of ester bonds and allow the synthesis of the ligand exclusively on alkaline-stable bonds. The method consists in activating the hydrophilic base resin, blocking the hydroxyl groups responsible for alkaline lability and performing the peptide synthesis exclusively via alkaline-stable amide bonds. Repeated cycles of IgG purification from a cell culture medium were performed, each followed by cleaning with aqueous NaOH (0.1 M, 0.5 M and 1 M). The IgG yield decreased from 91% to 85% after 200 purification cycles with 0.1 M NaOH. However, the IgG purity remained almost constant at around 95% based on SDS-PAGE analysis. The procedure presented is rapid, efficient and inexpensive and does not require any equipment other than the conventional instrumentation for peptide synthesis. The method also has a broad application since it is valid for any peptide ligand identified for the purification of a biopharmaceutical target.
提出了一种修饰树脂表面化学的策略，以生产亲水性肽基碱性稳定亲和吸附剂，用于从复杂介质中纯化生物制药。在这项工作中，以 HWRGWV-Toyopearl 树脂作为亲和吸附剂用于 IgG 的纯化为例。当直接在色谱基质上通过肽合成制备时，肽基树脂在碱性条件下不稳定。实际上，用 0.1 M NaOH 的水溶液再生会导致 40%的配体洗脱，导致 IgG 的产率从 85%下降到 23%。发现配体的洗脱是由大量通过碱性不稳定酯键偶联的肽引起的。设计了一种方法来防止酯键的形成，并允许仅通过碱性稳定的键合成配体。该方法包括激活亲水碱树脂，封闭导致碱性不稳定的羟基，并仅通过碱性稳定的酰胺键进行肽合成。从细胞培养基中进行了重复的 IgG 纯化循环，每次都用 0.1 M NaOH（0.5 M 和 1 M）进行清洗。在用 0.1 M NaOH 进行 200 次纯化循环后，IgG 的产率从 91%下降到 85%。然而，基于 SDS-PAGE 分析，IgG 的纯度几乎保持不变，约为 95%。所提出的方法快速、高效且廉价，不需要除肽合成常规仪器之外的任何设备。该方法还具有广泛的应用，因为它适用于为纯化任何生物制药目标而鉴定的任何肽配体。
