CHO细胞基因组中稳定hot spot位点研究进展

近些年来,治疗性重组蛋白类药物是生物制药领域研究的热点。工业化生产中常用于重组蛋白表达的细胞系是中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞。传统CHO细胞系的表达大多数基于随机整合的方式,这可能会使目标基因整合到异染色质区域或者不稳定的染色质区域,导致CHO细胞表达不稳定,需要多轮筛选才能获得理想的表达细胞系。最新研究表明,外源基因在CHO细胞预测/特定的基因组位点中进行特异性整合,可以使重组CHO细胞的表达保持长期一致性和稳定性。CHO细胞基因组中高效稳定的转录整合位点被称为热点(hot spot)。阐述CHO细胞基因组稳定的hot spot位点近几年的研究进展,其中包括热门的hot spot位点,以及如何研究新的hot spot位点的方法。总结如何将外源基因高效定位于预测的CHO细胞hot spot位点,实现高水平稳定的表达重组蛋白,为发现新的有效的hot spot位点,构建稳定表达CHO细胞系提供参考。     

外源蛋白在中国仓鼠卵巢细胞中高效表达的策略

高效表达外源蛋白,在生物制药中有重要意义.中国仓鼠卵巢细胞（Chinese hamster ovary cell）是表达外源蛋白的最佳真核表达系统之一.影响外源蛋白在CHO细胞表达的因素甚多,主要包括载体、宿主细胞和外源基因几方面.深入了解和灵活运用它们之间的关系,有助于获得外源基因在CHO细胞中的高效表达.     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因(k2tPA)、扩增基因(dhfr)、重组酶识别序列(FRT)及筛选基因(neo),且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO/dhfr-细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点(Hotspot)区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO/dhfr-细胞系。随后利用位点特异性重组系统(FLP-FRT)将外源基因定点整合到Hotspot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k2tPA的高表达,表达量达到17.1μg/106cell.24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

中国仓鼠卵巢细胞表达新技术

中国仓鼠卵巢细胞（CHO细胞）是基因工程药物生产的最佳表达系统之一,在生物制药中被广泛应用。传统的获得高表达CHO细胞株的方法费时、费力。近年来出现了一些CHO细胞高效表达新技术,它们从克服位置效应,提高基因转录效率、mRNA翻译效率及稳定性、筛选高表达细胞的效率等不同层次调控外源基因在CHO细胞中的高效表达。与MTX加压扩增基因获得高效表达外源基因的方法比较,能够节约时间、减少工作量,不易丢失高表达细胞株。     

在真核细胞表达制备重组人前蛋白转化酶枯草溶菌素9

分别优化、合成和构建了在毕赤酵母和哺乳动物细胞表达人前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)的PCSK9-p PICZαA和PCSK9-pcDNA4.0重组质粒,将重组质粒转化至GS115细胞和转染至中国仓鼠卵巢(chinese hamster ovary,CHO)细胞,诱导细胞表达包含his标签的PCSK9重组蛋白。通过SDS-PAGE电泳和Western-blot分析两种系统表达重组蛋白的能力,发现GS115表达的重组蛋白相对分子质量大于人天然PCSK9蛋白且容易发生降解,而CHO细胞能够高表达与人天然PCSK9蛋白相对分子质量相同的、稳定的、易于纯化的特异重组蛋白。采用His/Ni2+亲和层析柱纯化重组蛋白,获得纯度达90%以上的PCSK9重组蛋白;用纯化的重组蛋白免疫小鼠制备了高效价特异性PCSK9多克隆抗体。所建立的CHO细胞表达体系PCSK9的平均表达量为5.6 mg/L,为开发PCSK9抑制剂和深入研究PCSK9分子结构和功能奠定了基础。     

中国仓鼠卵巢细胞及其表达载体的改造

哺乳动物细胞因其表达的外源蛋白最接近天然构象,已成为生产重组蛋白药物的理想系统。其中,中国仓鼠卵巢细胞（CHO）是目前最为常用的表达系统,但这种系统也存在很多缺点,如大规模培养中表达量低、生产成本高、细胞无限度增殖及细胞凋亡等。目前,通过优化培养基配方和培养条件很难从根本上解决上述问题,必须从整个表达系统着手进行改造,其中CHO细胞本身和表达载体的改造最为关键。     

CHO/dhfr-细胞定点整合高效表达系统的建立

为了克服随机整合建立高表达细胞株时“位置效应”所带来的不可预知的后果,我们尝试建立基于定点整合的CHO高效表达系统。首先设计一个新的高效筛选载体pMCEscan。该载体含有报告基因（k2tPA）、扩增基因（dhfr）、重组酶识别序列（FRT）及筛选基因（neo）,且neo基因的表达经过系统的弱化,确保能够对基因组中的整合位点进行大规模的高效筛选。然后利用该载体转染CHO／dhfr^-细胞并进行大规模筛选以获得足够多的阳性克隆,并对阳性克隆进行系统分析,筛选出报告基因表达水平高、单拷贝且扩增效果好的克隆,此克隆被认为筛选载体整合入CHO细胞基因组中转录热点（Hotspot）区域,从而获得了能够实现外源基因在基因组中定点整合和有效表达的CHO／dhfr-细胞系。随后利用位点特异性重组系统（FLP-FRT）将外源基因定点整合到Hotspot区域,以实现外源基因在CHO细胞基因组中的定点整合及高效表达。并利用该细胞系实现了k2tPA的高表达,表达量达到17．1μg／10^6cell·24h。该研究致力于CHO细胞基因组中高表达位点的寻找和确认,建立基于定点整合的哺乳动物细胞高效表达系统。     

CHO细胞表达系统研究进展

CHO细胞表达系统是目前重组糖蛋白生产的首选系统。随着无血清悬浮培养技术、基因工程技术和大规模培养技术的应用和不断发展,CHO细胞表达系统已经成为生物技术药物最重要的表达或生产系统,并被广泛应用于抗体、重组蛋白药物和疫苗等产品的研发和生产中。近年来,针对CHO细胞表达系统在某些重组蛋白的表达和大规模生产中存在的不足,研究者们通过利用基因工程技术手段,结合重组蛋白表达机制的研究成果,为优化和应用CHO细胞表达系统做出了不懈努力。从培养基的优化、高产重组CHO细胞株的构建、大规模培养三个方面综述了CHO细胞表达系统的最近研究进展,以期为CHO细胞表达系统的研究与应用提供参考。     

APRT缺陷型CHO细胞系的建立及其重组蛋白表达能力评价北大核心CSCD

中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞是生产复杂重组药物蛋白的首选宿主细胞,腺嘌呤磷酸核糖转移酶(adenine phosphoribosyltransferase,APRT)催化腺嘌呤与磷酸核糖缩合形成腺苷一磷酸,是嘌呤生物合成步骤中的关键酶。采用基因编辑技术敲除CHO细胞中aprt基因,验证获得的APRT缺陷型CHO细胞系的生物学特性;构建两种真核表达载体:对照载体(含有目的基因增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和弱化载体(含有启动子和起始密码子突变的aprt弱化表达盒及EGFP),分别转染APRT缺陷型和野生型CHO细胞并筛选获得稳定转染的细胞池;重组CHO细胞传代培养60代并用流式细胞术检测EGFP表达的平均荧光强度,并比较不同实验组重组蛋白EGFP的表达稳定性。PCR扩增和测序结果表明,CHO细胞aprt基因成功敲除;获得的APRT缺陷型CHO细胞系在细胞形态、生长增殖、倍增时间等生物学特性方面与野生CHO细胞无显著差异。目的蛋白瞬时表达结果表明,与野生型CHO细胞相比,转染对照载体和弱化载体的APRT缺陷型CHO细胞系中EGFP的表达分别提高了42%±6%和56%±9%;特别是长期传代培养时,转染弱化载体的APRT缺陷型细胞中EGFP表达量显著高于野生型CHO细胞(P<0.05);构建的基于APRT缺陷型CHO细胞系能够明显提高重组蛋白的长期表达稳定性。研究结果为建立高效稳定的CHO细胞表达系统提供了一种有效的细胞工程策略。     

克服位置效应提高外源基因在CHO细胞中稳定高效表达的策略

能够生产有功用的治疗性蛋白的一个重要前提是获得稳定的重组蛋白高表达细胞株,然而筛选一个能够持续稳定表达外源蛋白的重组细胞株是费时费力的过程。有多篇文献报道了重组蛋白细胞株表达的不稳定性。位置效应是高表达细胞株不稳定性的重要因素,克服或利用位置效应是当前获得稳定高表达重组蛋白细胞株的有效途径。为解决外源基因插入的随机性所带来的不可预知的后果,可以事先在CHO细胞基因组中筛选转录热点区域,再通过位点特异性或同源重组的方式,实现外源基因的定点整合。各种调节位置效应的DNA元件陆续被发现,可以利用它们去调控基因表达及增加细胞株的稳定性。     

Endogenous BiP reporter system for simultaneous identification of ER stress and antibody production in Chinese hamster ovary cells

As the biopharmaceutical industry expands, improving the production of therapeutic proteins using Chinese hamster ovary (CHO) cells is important. However, excessive and complicated protein production causes protein misfolding and triggers endoplasmic reticulum (ER) stress. When ER stress occurs, cells mediate the unfolded protein response (UPR) pathway to restore protein homeostasis and folding capacity of the ER. However, when the cells fail to control prolonged ER stress, UPR induces apoptosis. Therefore, monitoring the degree of UPR is required to achieve high productivity and the desired quality. In this study, we developed a fluorescence-based UPR monitoring system for CHO cells. We integrated mGFP into endogenous HSPA5 encoding BiP, a major ER chaperone and the primary ER stress activation sensor, using CRISPR/Cas9-mediated targeted integration. The mGFP expression level changed according to the ER stress induced by chemical treatment and batch culture in the engineered cell line. Using this monitoring system, we demonstrated that host cells and recombinant CHO cell lines with different mean fluorescence intensities (MFI; basal expression levels of BiP) possess a distinct capacity for stress culture conditions induced by recombinant protein production. Antibody-producing recombinant CHO cell lines were generated using site-specific integration based on host cells equipped with the BiP reporter system. Targeted integrants showed a strong correlation between productivity and MFI, reflecting the potential of this monitoring system as a screening readout for high producers. Taken together, these data demonstrate the utility of the endogenous BiP reporter system for the detection of real-time dynamic changes in endogenous UPR and its potential for applications in recombinant protein production during CHO cell line development.     

Comparison of fluidized bed and ultrasonic cell-retention systems for high cell density mammalian cell culture

Economically viable biopharmaceutical production is to a high degree dependent on high product yields and stable fermentation systems that are easy to handle. In the current study we have compared two different fermentation systems for the production of recombinant protein from CHO cells. Both systems are fully scaleable and can be used for industrial high cell density bioprocesses. As a model cell line we have used a recombinant CHO cell line producing the enzyme arylsulfatase B (ASB). CHO cells were cultivated as adherent cell culture attached on Cytoline macroporous microcarrier (Amersham Biosciences, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR, Vogelbusch-Amersham Biosciences, Austria) and as suspension culture using a stirred tank bioreactor equipped with a BioSep ultrasonic resonator based cell separation device (Applikon, The Netherlands). Both systems are equally well-suited for stable, long-term high cell density perfusion cell culture and provide industrial scalability and high yields. For products such as the recombinant ASB, high perfusion rates and therefore short product bioreactor residence times may be of additional benefit.     

CRISPR/Cas9‐mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long‐term stability

CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a‐deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a‐deficent CHO cell line based on Dnmt3a KO displayed an enhanced long‐term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a‐deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a‐deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells.     

‘Omics driven discoveries of gene targets for apoptosis attenuation in CHO cells

Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical production. Improvements to cell lines and bioprocesses are constantly being explored. One of the major limitations of CHO cell culture is that the cells undergo apoptosis, leading to rapid cell death, which impedes reaching high recombinant protein titres. While several genetic engineering strategies have been successfully employed to reduce apoptosis, there is still room to further enhance CHO cell lines performance. ‘Omics analysis is a powerful tool to better understand different phenotypes and for the identification of gene targets for engineering. Here, we present a comprehensive review of previous CHO 'omics studies that revealed changes in the expression of apoptosis‐related genes. We highlight targets for genetic engineering that have reduced, or have the potential to reduce, apoptosis or to increase cell proliferation in CHO cells, with the final aim of increasing productivity.     

Proteomic studies support the use of multi-product immunoassays to monitor host cell protein impurities

In the biopharmaceutical industry, recombinant protein drugs are commonly produced in Chinese hamster ovary (CHO) cells. During the development process, removal of CHO cell-derived proteins from the biopharmaceutical product is monitored using multi-product immunoassays. Such immunoassays are developed by raising antibodies to a single CHO cell protein preparation. However, these assays are utilized to monitor CHO cell protein impurities during the recovery of products from different CHO cell lines. To address whether underlying differences between CHO cell lines result in sufficient protein expression changes to exclude the suitability of multi-product immunoassays, a comparative proteomics study of three independently generated CHO cell lines was performed. Statistical analysis of over 1000 proteins resolved by 2-D PAGE demonstrated that the protein expression profiles of three different CHO cell lines exhibit very few differences in protein expression. Only 11 qualitative changes in protein expression and 26 quantitative changes greater than two-fold were observed. Identification of protein spots by mass spectrometry revealed that many of the observed changes were due to post-translational modifications rather than expression of novel proteins in each cell line. These results suggest that multi-product immunoassays are suitable for monitoring host cell proteins in biopharmaceuticals produced in different CHO cell lines.     

Identification of CHO Endogenous Gene Regulatory Elements

The objective of this approach was to identify new CHO endogenous gene regulatory elements that are capable of regulating foreign gene expression in recombinant CHO host cells. The standard technology for the production of many biopharmaceutical products is frequently based on expression vectors that utilize strong mammalian viral promoters like SV40 or CMV which allow for very high expression rates but this may lead to constitutive over-expression resulting in a permanent stress for the cell. In addition, some heterologous promoters are cell-cycle dependent and can be subject to gene silencing generating heterogeneity within the cell population. Here, we describe the construction of a genomic CHO library and the subsequent identification and isolation of selected target sequences that are believed to be responsible for high level expression of the associated genes. The method that was used to isolate these regions of interest relies on gene specific amplification with primer pairs binding on different genes and the vector sequence. Flanking regions of these fragments were identified through Inverse PCR from fragmented and self-ligated genomic DNA. Expression levels of both the initially derived and the mapped fragments were determined through a luciferase reporter assay.     

Generation of recombinant CHO(dhfr-) cell lines by single selection for dhfr+ transformants.

In order to establish a mammalian cell expression system with a minimum of selection steps and a stable expression of microgram amounts of recombinant protein (human tissue-type plasminogen activator mutants and chimeric proteins) per 10(6) cells per day, we investigated Chinese hamster ovary cells and the dihydrofolate reductase-deficient Chinese hamster ovary cell line CHO(dhfr-). The 1tPA expression vector pCMVtPA was cotransfected either with the SV40 enhancer sequence containing dhfr expression vector pMT2 or with the enhancerless dhfr expression vector pAdD26SV(A) into CHO(dhfr-) cells. With both dhfr expression plasmids, selection for dhfr+ transformants followed by single dilution cloning was sufficient to generate cell lines with a production level of up to 4.6 micrograms tPA/10(6) cells.day. This approach is useful if gene amplification procedures are time-consuming and impracticable because of a large number of recombinant proteins. In order to establish CHO cell lines with a tPA expression level as high as that in the case of CHO(dhfr-) cells, repeated dilution cloning is necessary.     

Towards dynamic metabolic flux analysis in CHO cell cultures

Chinese hamster ovary (CHO) cells are the most widely used mammalian cell line for biopharmaceutical production, with a total global market approaching $100 billion per year. In the pharmaceutical industry CHO cells are grown in fed-batch culture, where cellular metabolism is characterized by high glucose and glutamine uptake rates combined with high rates of ammonium and lactate secretion. The metabolism of CHO cells changes dramatically during a fed-batch culture as the cells adapt to a changing environment and transition from exponential growth phase to stationary phase. Thus far, it has been challenging to study metabolic flux dynamics in CHO cell cultures using conventional metabolic flux analysis techniques that were developed for systems at metabolic steady state. In this paper we review progress on flux analysis in CHO cells and techniques for dynamic metabolic flux analysis. Application of these new tools may allow identification of intracellular metabolic bottlenecks at specific stages in CHO cell cultures and eventually lead to novel strategies for improving CHO cell metabolism and optimizing biopharmaceutical process performance.