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首页> 外文期刊>Biotechnology Progress >Fast Identification of Reliable Hosts for Targeted Cell Line Development from a Limited-Genome Screening Using Combined (pC31 Integrase and CRE-Lox Technologies
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Fast Identification of Reliable Hosts for Targeted Cell Line Development from a Limited-Genome Screening Using Combined (pC31 Integrase and CRE-Lox Technologies

机译：通过结合使用（pC31整合酶和CRE-Lox技术）从有限基因组筛选中快速鉴定目标细胞系开发的可靠宿主

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摘要

The use of targeted integration (TI) in cell line development (CLD) usually introduces one copy of a recombinant gene into a predetermined transcriptionally active locus. This reduces the heterogeneity typically associated with traditional random integration (RI) CLD with regards to varied productivity and instability, resulting from diverse chromosomal influences, varied copy numbers, and repeat-induced rearrangement. As such, TI CLD offers the hope of a predictable and consistent CLD process for establishing stable clones. However, given the low copy number, cell lines established from a TI CLD process tend to exhibit low productivity. Here, we describe our nonviral-based approach for quickly establishing and identifying TI hosts from a limited genome screening. Importantly, the TI hosts identified are consistent and reliable in supporting the production of diverse antibodies regardless of antibody subclass (IgG1 vs. IgG4) or prior traditional CLD performance (relatively easy vs. difficult to express antibodies). Moreover, an approximately twofold increase in titer can be achieved by using a CRE recombinase-mediated cassette exchange (RMCE) strategy with an exchange vector carrying two units of the antibody gene. Two RMCE hosts that were established were able to produce up to ~1.7 and 2 g/L of antibodies in nonoptimized fed-batch shake flask production cultures with chemically defined media. Potentially, this strategy may be applied to the production of bispecific antibodies with a fast turnaround time.
机译：在细胞系发育（CLD）中使用靶定整合（TI）通常会将重组基因的一个副本引入预定的转录活性基因座。这减少了通常与传统随机积分（RI）CLD相关的异质性，这些异质性是由各种染色体影响，不同的拷贝数和重复诱导的重排所导致的生产力和稳定性的变化。因此，TI CLD为建立稳定克隆提供了可预测且一致的CLD过程的希望。但是，由于拷贝数低，从TI CLD工艺建立的细胞系往往显示出低生产率。在这里，我们描述了我们基于非病毒的方法，用于通过有限的基因组筛选快速建立和识别TI宿主。重要的是，无论抗体亚类（IgG1与IgG4）还是先前的传统CLD性能（相对较难表达，相对较难表达），所鉴定的TI宿主在支持多种抗体的生产中都是一致且可靠的。而且，通过使用带有两个单位抗体基因的交换载体，使用CRE重组酶介导的盒交换（RMCE）策略，可以实现滴度的大约两倍增加。已建立的两个RMCE宿主能够在使用化学成分确定的培养基的非优化补料分批摇瓶生产培养物中产生高达〜1.7和2 g / L的抗体。潜在地，该策略可以应用于具有快速周转时间的双特异性抗体的产生。

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