发现并研究(GGGGS)n连接体的截断及其对哺乳动物细胞中表达的双特异性抗体生产率的影响。

IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Yan Fang, Xi Chen, Zhen Sun, Xiaodan Yan, Lani Shi, Congcong Jin
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引用次数: 0

摘要

蛋白质工程是设计或改造治疗蛋白质以增强疗效、提高安全性、降低免疫原性和改进给药方式的有力工具。融合蛋白是一类重要的治疗化合物,通常需要一个理想的连接体来结合不同的结构域以实现所需的功能。GGGGS [(G4S)n] 连接体因其灵活性和对蛋白酶的耐受性而常用于蛋白质工程。然而,在双特异性抗体的连接体中发现了意想不到的截断,这给生产和质量带来了挑战。本研究对含有 5*G4S 的双特异性抗体进行了研究,并确认了连接子的截断位置。我们的研究发现,密码子优化可以克服(G4S)n 连接子中高重复率和高 GC 含量的负面影响,可将截断率从 5-10% 降低到 1-5%。此外,在哺乳动物细胞中使用缩短的 3* 或 4*G4S 连接子时,截断的概率要比使用 5*G4S 连接子时低得多。在表达双特异性抗体的情况下,对含有较短 G4S 连接子的产品的生物活性和纯度进行了进一步研究和讨论。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Discovery and investigation of the truncation of the (GGGGS)n linker and its effect on the productivity of bispecific antibodies expressed in mammalian cells.

Protein engineering is a powerful tool for designing or modifying therapeutic proteins for enhanced efficacy, increased safety, reduced immunogenicity, and improved delivery. Fusion proteins are an important group of therapeutic compounds that often require an ideal linker to combine diverse domains to fulfill the desired function. GGGGS [(G4S)n] linkers are commonly used during the engineering of proteins because of their flexibility and resistance to proteases. However, unexpected truncation was observed in the linker of a bispecific antibody, which presented challenges in terms of production and quality. In this work, a bispecific antibody containing 5*G4S was investigated, and the truncation position of the linkers was confirmed. Our investigation revealed that codon optimization, which can overcome the negative influence of a high repetition rate and high GC content in the (G4S)n linker, may reduce the truncation rate from 5-10% to 1-5%. Moreover, the probability of truncation when a shortened 3* or 4*G4S linker was used was much lower than that when a 5*G4S linker was used in mammalian cells. In the case of expressing a bispecific antibody, the bioactivity and purity of the product containing a shorter G4S linker were further investigated and are discussed.

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来源期刊
Bioprocess and Biosystems Engineering
Bioprocess and Biosystems Engineering 工程技术-工程:化工
CiteScore
7.90
自引率
2.60%
发文量
147
审稿时长
2.6 months
期刊介绍: Bioprocess and Biosystems Engineering provides an international peer-reviewed forum to facilitate the discussion between engineering and biological science to find efficient solutions in the development and improvement of bioprocesses. The aim of the journal is to focus more attention on the multidisciplinary approaches for integrative bioprocess design. Of special interest are the rational manipulation of biosystems through metabolic engineering techniques to provide new biocatalysts as well as the model based design of bioprocesses (up-stream processing, bioreactor operation and downstream processing) that will lead to new and sustainable production processes. Contributions are targeted at new approaches for rational and evolutive design of cellular systems by taking into account the environment and constraints of technical production processes, integration of recombinant technology and process design, as well as new hybrid intersections such as bioinformatics and process systems engineering. Manuscripts concerning the design, simulation, experimental validation, control, and economic as well as ecological evaluation of novel processes using biosystems or parts thereof (e.g., enzymes, microorganisms, mammalian cells, plant cells, or tissue), their related products, or technical devices are also encouraged. The Editors will consider papers for publication based on novelty, their impact on biotechnological production and their contribution to the advancement of bioprocess and biosystems engineering science. Submission of papers dealing with routine aspects of bioprocess engineering (e.g., routine application of established methodologies, and description of established equipment) are discouraged.
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