Department of Chemical and Biological Engineering, University of Sheffield, Sheffield, UK.
Cell Line Development, BioTherapeutic Pharmaceutical Sciences, Pfizer Inc, Andover, Massachusetts.
Biotechnol Bioeng. 2018 Jun;115(6):1485-1498. doi: 10.1002/bit.26561. Epub 2018 Feb 26.
High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (<1/24,000 bases). Using a stable CHO transfectant pool harboring a randomly integrated 5 kB plasmid construct encoding GFP we found that 28% of recombinant plasmid copies contained at least one low frequency (<0.3%) point mutation. These mutations were predominantly found in GC base pairs (85%) and that there was no positional bias in mutation across the plasmid sequence. There was no discernable difference between the mutation frequencies of coding and non-coding DNA. The putative ratio of non-synonymous and synonymous changes within the open reading frames (ORFs) in the plasmid sequence indicates that natural selection does not impact upon the prevalence of these mutations. Here we have demonstrated the abundance of mutations that fall outside of the reported range of detection of next generation sequencing (NGS) and second generation sequencing (SGS) platforms, providing a methodology capable of being utilized in cell line development platforms to identify the fidelity of recombinant genes throughout the production process.
通过工程化的哺乳动物细胞培养物对生物编码重组 DNA 序列进行高保真复制，是开发用于生产生物疗法的稳定细胞系的必要前提。然而，与体内的哺乳动物细胞相比，永生化的哺乳动物细胞在整个基因组和特定基因座（热点）上都表现出更高的点突变频率。因此，重组 DNA 序列中可能会出现意想不到的突变，并在生产细胞群体中得以维持。这些突变可能会影响重组基因表达的稳定性，并导致具有可变生物活性和免疫原性的蛋白质序列变异体。因此，对重组 DNA 完整性进行严格的定量评估应成为细胞系开发过程的一部分，并且对于利用合成/多组分组装来工程化哺乳动物细胞的情况，例如评估重组 DNA 保真度或单一位点整合靶基因座的可变性，这是必不可少的质量保证指标。基于 Pacific Biosciences（加利福尼亚州门洛帕克）的单分子实时（SMRT）环状一致测序（CCS）技术，我们开发了一种 rDNA 序列分析工具，用于处理约 40,000 个单个重组 DNA 分子的多平行测序。对原始测序数据进行统计过滤后，我们证明该分析方法能够检测到 rDNA 中的单点突变，最低单突变频率为 0.0042%（<1/24,000 个碱基）。使用稳定的 CHO 转染池，该转染池含有随机整合的编码 GFP 的 5kb 质粒构建体，我们发现 28%的重组质粒拷贝至少含有一个低频率（<0.3%）的点突变。这些突变主要发生在 GC 碱基对（85%）中，并且在质粒序列中突变没有位置偏向性。编码和非编码 DNA 的突变频率没有明显差异。在质粒序列的开放阅读框（ORF）中同义和非同义变化的假定比例表明，自然选择不会影响这些突变的普遍性。在这里，我们已经证明了大量突变的存在，这些突变超出了下一代测序（NGS）和第二代测序（SGS）平台的检测范围，提供了一种能够在细胞系开发平台中使用的方法，以识别整个生产过程中重组基因的保真度。
