William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA.
Methods Mol Biol. 2023;2699:237-253. doi: 10.1007/978-1-0716-3362-5_13.
Glycoprotein therapeutics are currently used by large patient populations and generate significant revenue for the biopharmaceutical industry. These therapeutic proteins are currently purified at industrial scale using individualized processes involving multiple chromatographic steps. In the absence of a viable affinity platform method, the required chromatographic steps are difficult to develop and inevitably lead to significant yield losses. Further, during preclinical development, there is a need for reliable platform technologies capable of performing high-throughput screening for biologic candidates. Although affinity tags can provide a solution to some of these challenges, they require specific affinity resins, and the tag itself can interfere with the target protein characteristics. Fusion protein systems consisting of elastin-like polypeptide (ELP) and self-cleaving split inteins such as Npu DnaE can serve as potential non-chromatographic platform technologies for the single-step purification of tagless glycoproteins expressed in mammalian cells. In this chapter, we demonstrate the use of this technology to obtain highly purified anti-ErbB2 ML39 single-chain variable fragment (scFv) expressed from Expi293F suspension cells.
糖蛋白治疗药物目前被大量患者群体使用，并为生物制药行业带来了巨大的收入。这些治疗性蛋白目前在工业规模上使用个体化的过程进行纯化，其中涉及多个色谱步骤。在缺乏可行的亲和平台方法的情况下，所需的色谱步骤难以开发，并且不可避免地导致产量损失显著。此外，在临床前开发期间，需要能够进行高通量筛选生物候选物的可靠平台技术。尽管亲和标签可以为解决其中一些挑战提供解决方案，但它们需要特定的亲和树脂，并且标签本身可能会干扰目标蛋白的特性。由弹性蛋白样多肽 (ELP) 和自切割分裂内含肽（如 Npu DnaE）组成的融合蛋白系统可以作为一种潜在的非色谱平台技术，用于一步法纯化在哺乳动物细胞中表达的无标签糖蛋白。在本章中，我们展示了该技术在从悬浮的 Expi293F 细胞中表达的抗 ErbB2 ML39 单链可变片段 (scFv) 的高度纯化中的应用。
