细胞系开发代表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的哺乳动物宿主细胞系统。CHO细胞用于异源重组蛋白表达的主要应用在于将异位DNA稳定引入CHO宿主细胞基因组的相对简单性。自从CHO细胞在1980年代后期首次被用作生物制品工业生产的表达宿主以来,稳定的基因组转基因整合几乎完全是通过随机整合实现的。从那以后,由于缺乏可行的替代品,随机转基因整合已成为产生稳定的CHO生产细胞系的黄金标准。然而,最终证明,这种方法对细胞系发育过程提出了重大挑战,例如诱导细胞系不稳定的风险增加。近年来,新的和高度有效的(半)靶向转基因整合系统的重大发现为细胞系开发领域的技术革命铺平了道路。这些先进的方法包括转座酶的应用,重组酶或Cas9核酸酶介导的位点特异性基因组整合技术,这使得转基因表达盒能够无疤痕地转移到宿主细胞基因组内的转录活性基因座中。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,讨论了(半)目标集成技术的优点和局限性,并概述了生物制药行业的好处和机会。
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.