Quantifying orthogonality and separability: A method for optimizing resin selection and design
Quantifying orthogonality and separability: A method for optimizing resin selection and design
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量化正交性和可分离性：优化树脂选择和设计的方法
DOI：
10.1016/j.chroma.2020.461429
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发表时间：
2020
影响因子：
4.1
通讯作者：
Cramer, Steven M.
中科院分区：
文献类型：
--
作者：
Bilodeau, Camille L.;Vecchiarello, Nicholas A.;Altern, Scott;Cramer, Steven M.
最近的研究表明，通过组合正交的非亲和色谱步骤，可以快速开发出目标分子的有效纯化方法。在这里，我们建立在以前的工作，以开发一个灵活的框架，用于识别树脂，去除各种各样的产品的最佳正交杂质集。我们的方法包括筛选具有不同性质的蛋白质库（pI范围为5.0-11.4，通过HIC梯度中的保留测量不同的疏水性），并量化每个树脂分离库中每个蛋白质对的能力。然后将亲和性定义为两种树脂分离相互排斥的蛋白质对的程度。我们将这种方法应用于模型蛋白质库和一系列强的、耐盐的和多模式的离子交换剂，并评估哪些树脂组合表现良好，哪些表现不佳。特别地，我们发现强阳离子和强阴离子交换剂是正交的，而强和耐盐阴离子交换剂不是正交的。有趣的是，耐盐和多峰阳离子交换剂被发现是正交的，最好的树脂组合包括多峰阳离子交换树脂和触手阴离子交换树脂。这种量化正交性的方法是有价值的，因为它可以用作树脂设计以及工艺设计的标准。我们设想，使用该框架，将有可能设计一组下一代色谱配体，其被明确地工程化以优化分离性和正交性。
Recent studies have shown that by combining orthogonal, non-affinity chromatography steps, it is possible to rapidly develop efficient purification processes for molecules of interest. Here, we build upon previous work to develop a flexible framework for identifying resins that remove optimally orthogonal sets of impurities for a wide variety of products. Our approach involves screening a library of proteins with diverse properties (pI ranging from 5.0–11.4 and varying hydrophobicity measured by retention in a HIC gradient) on a library of resins and quantifying each resin’s ability to separate every protein pair in the library. Orthogonality is then defined as the degree to which two resins separate mutually exclusive sets of protein pairs. We applied this approach to a library of model proteins and a series of strong, salt tolerant, and multimodal ion exchangers and evaluated which resin combinations performed well and which performed poorly. In particular, we found that strong cation and strong anion exchangers were orthogonal, while strong and salt tolerant anion exchangers were not orthogonal. Interestingly, salt tolerant and multimodal cation exchangers were found to be orthogonal and the best resin combination included a multimodal cation exchange resin and a tentacular anion exchange resin. This approach for quantifying orthogonality is valuable in that it can be used both as a criteria for resin design as well as process design. We envision that, using this framework, it will be possible to design a set of next generation chromatography ligands that are explicitly engineered to optimize separability and orthogonality.