Combining regulated and constitutive protein expression significantly boosts protein expression by increasing productivity without affecting CHO cell growth

Chinese hamster ovary (CHO) cells are commonly used for the expression of therapeutic proteins. To increase the titer output of CHO production cultures either specific productivity (Qp), growth, or both need to be increased. Generally, Qp and growth are inversely correlated and cell lines with high Qp have slower growth and vice versa. During the cell line development (CLD) process, the faster-growing cells tend to take over the culture and represent the majority of the isolated clones post single cell cloning. In this study, combinations of regulated and constitutive expression systems were used to supertransfect targeted integration (TI) cell lines expressing the same antibody either constitutively or under-regulated expression. Clone screening with a hybrid expression system (inducible + constitutive) allowed identification and selection of higher titer clones under uninduced conditions, without a negative impact on cell growth during clone selection and expansion. Induction of the regulated promoter(s) during the production phase increased the Qp without negatively affecting growth, resulting in approximately twofold higher titers (from 3.5 to 6–7 g/L). This was also confirmed using a 2-site TI host where the gene of interest was expressed inducibly from Site 1 and constitutively from Site 2. Our findings suggest that such a hybrid expression CLD system can be used to increase production titers, providing a novel approach for expression of therapeutic proteins with high titer market demands.     

Utilizing a regulated targeted integration cell line development approach to systematically investigate what makes an antibody difficult to express

Chinese hamster ovary (CHO) cells are conventionally used to generate therapeutic cell lines via random integration (RI), where desired transgenes are stably integrated into the genome. Targeted integration (TI) approaches, which involve integration of a transgene into a specific locus in the genome, are increasingly utilized for CHO cell line development (CLD) in recent years. None of these CLD approaches, however, are suitable for expression of toxic or difficult-to-express molecules, or for determining the underlying causes for poor expression of some molecules. Here we introduce a regulated target integration (RTI) system, where the desired transgene is integrated into a specific locus and transcribed under a regulated promoter. This system was used to determine the underlying causes of low protein expression for a difficult-to-express antibody (mAb-A). Interestingly, we observed that both antibody heavy chain (HC) and light chain (LC) subunits of mAb-A independently contributed to its low expression. Analysis of RTI cell lines also revealed that while mAb-A LC triggered accumulation of intracellular BiP, its HC displayed impaired degradation and clearance. RTI pools, generated by swapping the WT or point-mutant versions of difficult-to-express antibody HC and LC with that of an average antibody, were instrumental in understanding the contribution of HC and LC subunits to the overall antibody expression. The ability to selectively turn off the expression of a target transgene in an RTI system could help to directly link expression of a transgene to an observed adverse effect. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2772, 2019.     

Utilizing targeted integration CHO pools to potentially accelerate the GMP manufacturing of monoclonal and bispecific antibodies

Monoclonal antibodies (mAbs) are effective therapeutic agents against many acute infectious diseases including COVID-19, Ebola, RSV, Clostridium difficile, and Anthrax. mAbs can therefore help combat a future pandemic. Unfortunately, mAb development typically takes years, limiting its potential to save lives during a pandemic. Therefore “pandemic mAb” timelines need to be shortened. One acceleration tool is “deferred cloning” and leverages new Chinese hamster ovary (CHO) technology based on targeted gene integration (TI). CHO pools, instead of CHO clones, can be used for Phase I/II clinical material production. A final CHO clone (producing the mAb with a similar product quality profile and preferably with a higher titer) can then be used for Phase III trials and commercial manufacturing. This substitution reduces timelines by ~3 months. We evaluated our novel CHO TI platform to enable deferred cloning. We created four unique CHO pools expressing three unique mAbs (mAb1, mAb2, and mAb3), and a bispecific mAb (BsAb1). We then performed single-cell cloning for mAb1 and mAb2, identifying three high-expressing clones from each pool. CHO pools and clones were inoculated side-by-side in ambr15 bioreactors. CHO pools yielded mAb titers as high as 10.4 g/L (mAb3) and 7.1 g/L (BsAb1). Subcloning yielded CHO clones expressing higher titers relative to the CHO pools while yielding similar product quality profiles. Finally, we showed that CHO TI pools were stable by performing a 3-month cell aging study. In summary, our CHO TI platform can increase the speed to clinic for a future “pandemic mAb.”     

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。     

It's time to regulate: Coping with product‐induced nongenetic clonal instability in CHO cell lines via regulated protein expression

Clonal instability and titer loss during Chinese hamster ovary (CHO) cell line development (CLD) has several underlying causes, the most prominent of which are DNA copy number loss and DNA silencing. However, in some cases, clonal instability is due to the toxicity of the therapeutic protein(s) that clones express. Unlike DNA copy number loss, which may occur in some clones or DNA silencing that is prevalent in certain regions of the genome, the hallmark of product induced clonal instability is its manifestation in all the selected clones. To circumvent such product induced clonal instability, we have developed a vector construct that utilizes a regulated protein expression system in which the constitutive expression of the target protein(s) is prevented unless doxycycline is added to the culture. We have then successfully used this system to express, at high titers, an antibody for which constitutive expression results in clonal instability perhaps due to intracellular accumulation of the antibody. Our data shows that unlike the constitutively expressed or continuously induced clones, uninduced clones do not display instability. Furthermore, maintaining the uninduced clones in culture for months or subjecting them to freeze‐thaws did not have any effects on their titers. All together, our findings suggest that a regulated expression system could be suitable for production of difficult proteins that trigger instability. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1432–1440, 2014     

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

In recent years, coherent with growing biologics portfolios also the number of complex and thus difficult‐to‐express (DTE) therapeutic proteins has increased considerably. DTE proteins challenge bioprocess development and can include various therapeutic protein formats such as monoclonal antibodies (mAbs), multi‐specific affinity scaffolds (e.g., bispecific antibodies), cytokines, or fusion proteins. Hence, the availability of robust and versatile Chinese hamster ovary (CHO) host cell factories is fundamental for high‐yielding bioprocesses. MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve process performance of CHO manufacturing cell lines. However, there has not been any report demonstrating the impact of beneficial miRNAs on industrial cell line development (CLD) yet. To address this question, we established novel CHO host cells constitutively expressing a pro‐productive miRNA: miR‐557. Novel host cells were tested in two independent CLD campaigns using two different mAb candidates including a normal as well as a DTE antibody. Presence of miR‐557 significantly enhanced each process step during CLD in a product independent manner. Stable expression of miR‐557 increased the probability to identify high‐producing cell clones. Furthermore, production cell lines derived from miR‐557 expressing host cells exhibited significantly increased final product yields in fed‐batch cultivation processes without compromising product quality. Strikingly, cells co‐expressing miR‐557 and a DTE antibody achieved a twofold increase in product titer compared to clones co‐expressing a negative control miRNA. Thus, host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial CLD especially with regard to DTE proteins. Biotechnol. Bioeng. 2017;114: 1495–1510. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Optimized CRISPR/Cas9 strategy for homology-directed multiple targeted integration of transgenes in CHO cells

Site-specific integration has emerged as a promising strategy for precise Chinese hamster ovary (CHO) cell line engineering and predictable cell line development (CLD). CRISPR/Cas9 with the homology-directed repair (HDR) pathway enables precise integration of transgenes into target genomic sites. However, inherent recalcitrance to HDR-mediated targeted integration (TI) of transgenes results in low targeting efficiency, thus requiring a selection process to find a targeted integrant in CHO cells. Here, we explored several parameters that influence the targeting efficiency using a promoter-trap-based single- or double-knock-in (KI) monitoring system. A simple change in the donor template design by the addition of single-guide RNA recognition sequences strongly increased KI efficiency (2.9–36.0 fold), depending on integration sites and cell culture mode, compared to conventional circular donor plasmids. Furthermore, sequential and simultaneous KI strategies enabled us to obtain populations with ~1–4% of double-KI cells without additional enrichment procedures. Thus, this simple optimized strategy not only allows efficient CRISPR/Cas9-mediated TI in CHO cells but also paves the way for the applicability of multiplexed KIs in one experimental step without the need for sequential and independent CHO–CLD procedures.     

Determination of the baculovirus transducing titer in mammalian cells

Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol to evaluate the transducing titers of baculovirus stocks. The virus was diluted twofold serially and used to transduce HeLa cells. The resultant transduction efficiencies were measured by flow cytometry for the calculation of transducing titers. Compared to the infectious titer, the determination of transducing titer is more reproducible as the standard deviations among measurements are smaller. Also, the transducing titers can be obtained in 24 h, which is significantly faster as opposed to 4-7 days to obtain the infectious titer. More importantly, we demonstrated that baculoviruses with higher transducing titers could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the transducing titer was representative of the baculovirus transducing ability. This finding is particularly significant for ex vivo gene delivery whereby unconcentrated viruses are used for transduction and long-term transgene expression is desired. In this regard, our titration protocol provides a simple, fast, and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments.     

Slashing the timelines: Opting to generate high‐titer clonal lines faster via viability‐based single cell sorting

Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 ? 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry‐based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:198–207, 2016     

Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.     

Enhanced CHO Clone Screening: Application of Targeted Locus Amplification and Next‐Generation Sequencing Technologies for Cell Line Development

Early analytical clone screening is important during Chinese hamster ovary (CHO) cell line development of biotherapeutic proteins to select a clonally derived cell line with most favorable stability and product quality. Sensitive sequence confirmation methods using mass spectrometry have limitations in throughput and turnaround time. Next‐generation sequencing (NGS) technologies emerged as alternatives for CHO clone analytics. We report an efficient NGS workflow applying the targeted locus amplification (TLA) strategy for genomic screening of antibody expressing CHO clones. In contrast to previously reported RNA sequencing approaches, TLA allows for targeted sequencing of genomic integrated transgenic DNA without prior locus information, robust detection of single‐nucleotide variants (SNVs) and transgenic rearrangements. During clone selection, TLA/NGS revealed CHO clones with high‐level SNVs within the antibody gene and we report in another case the utility of TLA/NGS to identify rearrangements at transgenic DNA level. We also determined detection limits for SNVs calling and the potential to identify clone contaminations by TLA/NGS. TLA/NGS also allows to identify genetically identical clones. In summary, we demonstrate that TLA/NGS is a robust screening method useful for routine clone analytics during cell line development with the potential to process up to 24 CHO clones in less than 7 workdays.     

Structural basis for interactions between tenascins and lectican C-type lectin domains: evidence for a crosslinking role for tenascins

The C-terminal G3 domains of lecticans mediate crosslinking to diverse extracellular matrix (ECM) proteins during ECM assembly, through their C-type lectin (CLD) subdomains. The structure of the rat aggrecan CLD in a Ca(2+)-dependent complex with fibronectin type III repeats 3-5 of rat tenascin-R provides detailed support for such crosslinking. The CLD loops bind Ca2+ like other CLDs, but no carbohydrate binding is observed or possible. This is thus the first example of a direct Ca(2+)-dependent protein-protein interaction of a CLD. Surprisingly, tenascin-R does not coordinate the Ca2+ ions directly. Electron microscopy confirms that full-length tenascin-R and tenascin-C crosslink hyaluronan-aggrecan complexes. The results are significant for the binding of all lectican CLDs to tenascin-R and tenascin-C. Comparison of the protein interaction surface with that of P-selectin in complex with the PGSL-1 peptide suggests that direct protein-protein interactions of Ca(2+)-binding CLDs may be more widespread than previously appreciated.     

The EF‐1α promoter maintains high‐level transgene expression from episomal vectors in transfected CHO‐K1 cells

In our previous study, we demonstrated that episomal vectors based on the characteristic sequence of matrix attachment regions (MARs) and containing the cytomegalovirus (CMV) promoter allow transgenes to be maintained episomally in Chinese hamster ovary (CHO) cells. However, the transgene expression was unstable and the number of copies was low. In this study, we focused on enhancers, various promoters and promoter variants that could improve the transgene expression stability, expression magnitude (level) and the copy number of a MAR‐based episomal vector in CHO‐K1 cells. In comparison with the CMV promoter, the eukaryotic translation elongation factor 1 α (EF‐1α, gene symbol EEF1A1) promoter increased the transfection efficiency, the transgene expression, the proportion of expression‐positive clones and the copy number of the episomal vector in long‐term culture. By contrast, no significant positive effects were observed with an enhancer, CMV promoter variants or CAG promoter in the episomal vector in long‐term culture. Moreover, the high‐expression clones harbouring the EF‐1α promoter tended to be more stable in long‐term culture, even in the absence of selection pressure. According to these findings, we concluded that the EF‐1α promoter is a potent regulatory sequence for episomal vectors because it maintains high transgene expression, transgene stability and copy number. These results provide valuable information on improvement of transgene stability and the copy number of episomal vectors.     

Use of Flp-mediated cassette exchange in the development of a CHO cell line stably producing erythropoietin

The feasibility of the use of Flp-mediated cassette exchange in the development of a CHO cell line, which produces erythropoietin (EPO) stably and largely, was investigated. A stable, high enhanced green fluorescence protein (EGFP)-producing clone was screened by extensive flow cytometric analysis. An EPO expression unit was targeted into the premarked locus of the stable parental clone by Flp-mediated cassette exchange and a correctly targeted clone (FC28T7) was obtained. The EPO production of FC28T7 was proven to be stable in long-term culture. Furthermore, the Flp-mediated cassette exchange did not alter the stable parental clone's characteristics concerning transgene expression level and stability. Taken together, the data obtained here indicated that the establishment of CHO cell lines stably producing a desired protein is achievable using Flp-mediated cassette exchange.     

Overexpression of mutant cell division cycle 25 homolog B (CDC25B) enhances the efficiency of selection in Chinese hamster ovary cells

The effects of mutant cell division cycle 25 homolog B (CDC25B) overexpression on the generation of cells producing a monoclonal antibody were investigated in Chinese hamster ovary (CHO) cells. Mutant CDC25B (m-CDC25B) expression plasmids were transfected into CHO DG44-derived cells producing a monoclonal antibody, and the frequency of highly producing cells was assessed following gene amplification in the presence of 250 nM methotrexate. Most of the clones obtained from the m-CDC25B-overexpressing cells had higher antibody titers than did mock-transfected control cells. This arose from either higher transgene copy numbers or higher mRNA expression levels for the antibody. However, the high mRNA expression levels were not always accompanied by increases in transgene copy numbers. Our results suggest that cells producing high levels of a monoclonal antibody can be selected efficiently using m-CDC25B overexpression.     

A novel regulatory element (E77) isolated from CHO‐K1 genomic DNA enhances stable gene expression in Chinese hamster ovary cells

Vectors flanked by regulatory DNA elements have been used to generate stable cell lines with high productivity and transgene stability; however, regulatory elements in Chinese hamster ovary (CHO) cells, which are the most widely used mammalian cells in biopharmaceutical production, are still poorly understood. We isolated a novel gene regulatory element from CHO‐K1 cells, designated E77, which was found to enhance the stable expression of a transgene. A genomic library was constructed by combining CHO‐K1 genomic DNA fragments with a CMV promoter‐driven GFP expression vector, and the E77 element was isolated by screening. The incorporation of the E77 regulatory element resulted in the generation of an increased number of clones with high expression, thereby enhancing the expression level of the transgene in the stable transfectant cell pool. Interestingly, the E77 element was found to consist of two distinct fragments derived from different locations in the CHO genome shotgun sequence. High and stable transgene expression was obtained in transfected CHO cells by combining these fragments. Additionally, the function of E77 was found to be dependent on its site of insertion and specific orientation in the vector construct. Our findings demonstrate that stable gene expression mediated by the CMV promoter in CHO cells may be improved by the isolated novel gene regulatory element E77 identified in the present study.