Goodarzi Maryam Moazami, Sardari Soroush, Kheirkhah Omolbani
Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, Karaj, Iran; Drug Design and Bioinformatics Unit, Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
Drug Design and Bioinformatics Unit, Department of Medical Biotechnology, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
J Chromatogr A. 2025 Aug 30;1757:466134. doi: 10.1016/j.chroma.2025.466134. Epub 2025 Jun 10.
Multimodal or mixed-mode chromatography (MMC) resins are promising for pharmaceutical recombinant proteins due to the potential for developing orthogonal purification steps with unique selectivity. However, the complex behavior of multimodal ligands makes them difficult to use. Generally, several factors, including ligand design (functional groups, spacer/ligand length and flexibility, ligand orientation), ligand density and distribution, and using optimal operational conditions (elution strategy and composition), define the final selectivity of the purification process. Adjusting multimodal interactions results in modified binding strength and adsorption. In some cases, optimizing the interaction potential of each modality in the structure of the multimodal ligand could achieve the desired property, while others show contrasting results. The ideal linker length between various moieties improves retention in some cases. A high adsorption rate and selectivity have been achieved respectively, with a uniform and heterogeneous distribution of ligand surface. However, the result of functional group variation on resin behavior varied based on protein characteristics. Moreover, control over operational conditions like pH, salt concentration, temperature, additives, strategies for protein purification, and displacers is essential for producing selective purification steps using these resins. Nevertheless, a deep understanding of the molecular interactions between ligands and the target biomolecules during binding and elution processes provides superior assistance for exploiting the selectivity of these resins by selecting an optimal purification strategy, ligand type, and ligand design. For this purpose, various experimental and modeling tools (mechanistic or empirical) have been used for the successful study of the chromatographic process. Although all of these methods have their potential and limitations, using them provides a beneficial understanding of the purification process. An inclusive overview of the mechanisms and methods for exploiting the selectivity and orthogonality of MMC resins is a gap that is being addressed in this review for further successful purification studies.
多模式或混合模式色谱（MMC）树脂对于药物重组蛋白很有前景，因为有可能开发出具有独特选择性的正交纯化步骤。然而，多模式配体的复杂行为使其难以使用。一般来说，几个因素，包括配体设计（官能团、间隔基/配体长度和柔韧性、配体取向）、配体密度和分布，以及使用最佳操作条件（洗脱策略和组成），决定了纯化过程的最终选择性。调整多模式相互作用会导致结合强度和吸附的改变。在某些情况下，优化多模式配体结构中每种模式的相互作用潜力可以实现所需的性质，而其他情况则显示出相反的结果。在某些情况下，不同部分之间理想的连接子长度可提高保留率。分别实现了高吸附率和选择性，配体表面分布均匀且不均匀。然而，官能团变化对树脂行为的影响因蛋白质特性而异。此外，控制pH、盐浓度、温度、添加剂、蛋白质纯化策略和置换剂等操作条件对于使用这些树脂产生选择性纯化步骤至关重要。尽管如此，深入了解配体与目标生物分子在结合和洗脱过程中的分子相互作用，通过选择最佳纯化策略、配体类型和配体设计，为利用这些树脂的选择性提供了卓越的帮助。为此，已使用各种实验和建模工具（机理或经验）来成功研究色谱过程。尽管所有这些方法都有其潜力和局限性，但使用它们有助于对纯化过程有有益的理解。对利用MMC树脂选择性和正交性的机制和方法进行全面概述，是本综述中正在解决的一个空白，以便进行进一步的成功纯化研究。
