Chusainow Janet, Yang Yuan Sheng, Yeo Jessna H M, Toh Poh Choo, Asvadi Parisa, Wong Niki S C, Yap Miranda G S
Bioprocessing Technology Institute, A*STAR Biomedical Sciences Institutes, 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.
Biotechnol Bioeng. 2009 Mar 1;102(4):1182-96. doi: 10.1002/bit.22158.
Generating stable, high-producing cell lines for recombinant protein production requires an understanding of the potential limitations in the cellular machinery for protein expression. In order to increase our understanding of what makes a stable high producer, we have generated a panel of 17 recombinant monoclonal antibody expressing Chinese hamster ovary subclones (CHO-mAb) with specific productivities ranging between 3 and 75 pg cell(-1) day(-1) using the dihydrofolate reductase (dhfr) expression system and compared the molecular features of these high- and low-producer clones. The relative heavy chain (HC) and light chain (LC) transgene copy numbers and mRNA levels were determined using real-time quantitative PCR (RT qPCR). We observed that not only higher transgene copy numbers and mRNA levels of both HC and LC were characteristic for the high-producer clones as compared to the low-producer clones but also a more favorable HC to LC transgene copy numbers ratio. By studying the long-term stability of the CHO-mAb subclones in the absence of methotrexate (MTX) selective pressure over 36 passages we observed a 35-92% decrease in volumetric productivity, primarily caused by a significant decrease in HC and LC mRNA levels with little change in the transgene copy numbers. Using Southern blot hybridization we analyzed the HC and LC transgene integration patterns in the host chromosome and their changes in course of gene amplification and long-term culturing. We observed that MTX-induced gene amplification caused chromosomal rearrangements resulting in clonal variability in regards to growth, productivity, and stability. No further obvious DNA rearrangements occurred during long-term culturing in the absence of MTX, indicating that other mechanisms were responsible for the decreased transcription efficiency. Our results implicate that the amplified transgene sequences were arranged in tandem repeats potentially triggering repeat-induced gene silencing. We hypothesize that the decline in transgene mRNA levels upon long-term culturing without MTX was mainly caused by transgene silencing consequently leading to a loss in mAb productivity. The exact molecular mechanisms causing production instability are not yet fully understood. The herein described extensive characterization studies could help understand the limitations to high-level, stable recombinant protein production and find ways to improving and accelerating the process for high-producer cell line generation and selection.
生成用于重组蛋白生产的稳定高产细胞系需要了解蛋白质表达细胞机制中的潜在限制。为了增进我们对稳定高产细胞系构成因素的理解，我们利用二氢叶酸还原酶（dhfr）表达系统构建了一组17个表达重组单克隆抗体的中国仓鼠卵巢亚克隆（CHO-mAb），其比生产率在3至75 pg细胞⁻¹天⁻¹之间，并比较了这些高产和低产克隆的分子特征。使用实时定量PCR（RT qPCR）测定了相对重链（HC）和轻链（LC）转基因拷贝数及mRNA水平。我们观察到，与低产克隆相比，高产克隆不仅具有更高的HC和LC转基因拷贝数及mRNA水平，而且HC与LC转基因拷贝数的比例更有利。通过研究CHO-mAb亚克隆在无甲氨蝶呤（MTX）选择压力下36代的长期稳定性，我们观察到体积生产率下降了35 - 92%，这主要是由于HC和LC mRNA水平显著下降，而转基因拷贝数变化不大。我们使用Southern印迹杂交分析了HC和LC转基因在宿主染色体中的整合模式及其在基因扩增和长期培养过程中的变化。我们观察到MTX诱导的基因扩增导致染色体重排，从而在生长、生产率和稳定性方面产生克隆变异性。在无MTX的长期培养过程中未发生进一步明显的DNA重排，表明其他机制导致了转录效率的降低。我们的结果表明，扩增的转基因序列以串联重复形式排列，可能触发重复诱导的基因沉默。我们推测，在无MTX的长期培养中转基因mRNA水平的下降主要是由转基因沉默导致的，从而导致单克隆抗体生产率的损失。导致生产不稳定的确切分子机制尚未完全了解。本文所述的广泛表征研究有助于理解高水平、稳定重组蛋白生产的限制，并找到改进和加速高产细胞系生成和选择过程的方法。
