Increasing attention has been paid to the purity of therapeutic proteins imposing extensive costs and challenges to the downstream processing of biopharmaceuticals. One of the efforts, that has been exerted to overcome such limitations, was developing multimodal or mixed-mode chromatography (MMC) resins for launching selective, orthogonal, non-affinity purification platforms. Despite relatively extensive usage of MMC resins, their real potential and fulfillment have not been extensively reviewed yet. In this work, the explanation of practical and key aspects of downstream processing of recombinant proteins with or without MMC resins was debated, as being useful for further purification process development. This review has been written as a step-by-step guide to deconvolute both inherent protein purification and MMC complexities. Here, after complete elucidation of the potential of MMC resins, the effects of frequently used additives (mobile phase modifiers) and their possible interactions during the purification process, the critical characteristics of common product-related impurities (e.g., aggregates, charge variants, fragments), host-related impurities (e.g., host cell protein and DNA) and process related impurities (e.g., endotoxin, and viruses) with solved or unsolved challenges of traditional and MMC resins have been discussed. Such collective experiences which are reported in this study could be considered as an applied guide for developing successful downstream processing in challenging conditions by providing a clear insight into complex MMC resins and impurities.

中文翻译：

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清晰了解复杂的多模式填料和杂质，以克服重组蛋白纯化挑战：综述


治疗性蛋白质的纯度越来越受到关注，这给生物制药的下游加工带来了巨大的成本和挑战。为克服这些限制而付出的努力之一是开发多模式或混合模式层析 （MMC） 填料，以推出选择性、正交、非亲和纯化平台。尽管 MMC 树脂的使用相对广泛，但其真正的潜力和实现尚未得到广泛审查。在这项工作中，对使用或不使用 MMC 填料的重组蛋白下游加工的实际和关键方面的解释进行了辩论，认为这些都有助于进一步的纯化工艺开发。本综述是作为去复杂化固有蛋白质纯化和 MMC 复杂性的分步指南而编写的。本文中，在完全阐明了 MMC 填料的潜力、常用添加剂（流动相改性剂）的影响及其在纯化过程中可能的相互作用之后，常见产品相关杂质（如聚集体、电荷异构体、片段）、宿主相关杂质（如宿主细胞蛋白和 DNA）和工艺相关杂质（如内毒素和病毒）的关键特性与传统填料和 MMC 填料已解决或未解决的挑战被讨论过。本研究中报告的这些集体经验可以被视为在具有挑战性的条件下开发成功的下游加工的应用指南，因为它清楚地了解了复杂的 MMC 树脂和杂质。