Cell Line Development, Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH and Co.KG, Biberach an der Riss, Germany.
Cell Line Development, Bioprocess Development Biologicals, Boehringer Ingelheim Pharma GmbH and Co.KG, Biberach an der Riss, Germany.
Biotechnol Adv. 2024 Oct;75:108402. doi: 10.1016/j.biotechadv.2024.108402. Epub 2024 Jun 29.
Cell line development represents a crucial step in the development process of a therapeutic glycoprotein. Chinese hamster ovary (CHO) cells are the most frequently employed mammalian host cell system for the industrial manufacturing of biologics. The predominant application of CHO cells for heterologous recombinant protein expression lies in the relative simplicity of stably introducing ectopic DNA into the CHO host cell genome. Since CHO cells were first used as expression host for the industrial production of biologics in the late 1980s, stable genomic transgene integration has been achieved almost exclusively by random integration. Since then, random transgene integration had become the gold standard for generating stable CHO production cell lines due to a lack of viable alternatives. However, it was eventually demonstrated that this approach poses significant challenges on the cell line development process such as an increased risk of inducing cell line instability. In recent years, significant discoveries of new and highly potent (semi)-targeted transgene integration systems have paved the way for a technological revolution in the cell line development sector. These advanced methodologies comprise the application of transposase-, recombinase- or Cas9 nuclease-mediated site-specific genomic integration techniques, which enable a scarless transfer of the transgene expression cassette into transcriptionally active loci within the host cell genome. This review summarizes recent advancements in the field of transgene integration technologies for CHO cell line development and compare them to the established random integration approach. Moreover, advantages and limitations of (semi)-targeted integration techniques are discussed, and benefits and opportunities for the biopharmaceutical industry are outlined.
细胞系开发是治疗性糖蛋白开发过程中的关键步骤。中国仓鼠卵巢（CHO）细胞是用于生物制品工业制造的最常用哺乳动物宿主细胞系。CHO 细胞用于异源重组蛋白表达的主要应用在于将外源 DNA 稳定引入 CHO 宿主细胞基因组相对简单。自 20 世纪 80 年代末 CHO 细胞首次被用作生物制品的工业生产表达宿主以来，稳定的基因组转基因整合几乎完全通过随机整合实现。此后，由于缺乏可行的替代方法，随机转基因整合成为生成稳定 CHO 生产细胞系的金标准。然而，最终证明这种方法在细胞系开发过程中存在重大挑战，例如增加了诱导细胞系不稳定的风险。近年来，新型高效（半）靶向转基因整合系统的重大发现为细胞系开发领域带来了技术革命。这些先进的方法包括转座酶、重组酶或 Cas9 核酸酶介导的靶向基因组整合技术的应用，可实现转基因表达盒无疤痕地转移到宿主细胞基因组中转录活性位点。本文综述了 CHO 细胞系开发中转基因整合技术的最新进展，并将其与已建立的随机整合方法进行了比较。此外，还讨论了（半）靶向整合技术的优缺点，并概述了它们对生物制药行业的益处和机遇。
