Chinese hamster ovary (CHO) cells are the most widely used production host for therapeutic proteins. With the recent emergence of CHO genome sequences, CHO cell line engineering has taken on a new ...aspect through targeted genome editing. The bacterial clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9) system enables rapid, easy and efficient engineering of mammalian genomes. It has a wide range of applications from modification of individual genes to genome‐wide screening or regulation of genes. Facile genome editing using CRISPR/Cas9 empowers researchers in the CHO community to elucidate the mechanistic basis behind high level production of proteins and product quality attributes of interest. In this review, we describe the basis of CRISPR/Cas9‐mediated genome editing and its application for development of next generation CHO cell factories while highlighting both future perspectives and challenges. As one of the main drivers for the CHO systems biology era, genome engineering with CRISPR/Cas9 will pave the way for rational design of CHO cell factories.
With the recent emergence of CHO genomic sequences and the RNA‐guided CRISPR/Cas9 technology, CHO cell line engineering has entered a new era with efficient targeted genome engineering. In this review the authors describe the basis for CRISPR/Cas9‐mediated genome engineering and its applications, which will pave the way for rational design of the next generation of CHO cell factories.
Beyond potency, a good developability profile is a key attribute of a biological drug. Selecting and screening for such attributes early in the drug development process can save resources and avoid ...costly late-stage failures. Here, we review some of the most important developability properties that can be assessed early on for biologics. These include the influence of the source of the biologic, its biophysical and pharmacokinetic properties, and how well it can be expressed recombinantly. We furthermore present in silico, in vitro, and in vivo methods and techniques that can be exploited at different stages of the discovery process to identify molecules with liabilities and thereby facilitate the selection of the most optimal drug leads. Finally, we reflect on the most relevant developability parameters for injectable versus orally delivered biologics and provide an outlook toward what general trends are expected to rise in the development of biologics.
Many branches of biology depend on stable and predictable recombinant gene expression, which has been achieved in recent years through targeted integration of the recombinant gene into defined ...integration sites. However, transcriptional levels of recombinant genes in characterized integration sites are controlled by multiple components of the integrated expression cassette. Lack of readily available tools has inhibited meaningful experimental investigation of the interplay between the integration site and the expression cassette components. Here we show in a systematic manner how multiple components contribute to final net expression of recombinant genes in a characterized integration site. We develop a CRISPR/Cas9-based toolbox for construction of mammalian cell lines with targeted integration of a landing pad, containing a recombinant gene under defined 5′ proximal regulatory elements. Generated site-specific recombinant cell lines can be used in a streamlined recombinase-mediated cassette exchange for fast screening of different expression cassettes. Using the developed toolbox, we show that different 5′ proximal regulatory elements generate distinct and robust recombinant gene expression patterns in defined integration sites of CHO cells with a wide range of transcriptional outputs. This approach facilitates the generation of user-defined and product-specific gene expression patterns for programmable mammalian cell engineering.
Pooled CRISPR screens have been widely applied to mammalian and other organisms to elucidate the interplay between genes and phenotypes of interest. The most popular method for delivering the CRISPR ...components into mammalian cells is lentivirus based. However, because lentivirus is not always an option, virus-free protocols are starting to emerge. Here, we demonstrate an improved virus-free, genome-wide CRISPR screening platform for Chinese hamster ovary cells with 75,488 gRNAs targeting 15,028 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad, resulting in a very high percentage of single gRNA insertions and minimal clonal variation. Using this platform, we perform a negative selection screen on cell proliferation that identifies 1,980 genes that affect proliferation and a positive selection screen on the toxic endoplasmic reticulum stress inducer, tunicamycin, that identifies 77 gene knockouts that improve survivability.
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•A recombinase-based method for virus-free, genome-wide CRISPR knockout screens•Utilizes the same libraries and analysis tools as traditional virus-based screens•Avoids noise associated with virus-based random insertions into the genome
Although lentivirus-based delivery of genome-wide CRISPR screen components has proven successful, there are situations in, e.g., industry and hospitals where working with live viruses is difficult or simply not an option. For those situations we have developed an alternative to virus-based, genome-wide CRISPR screens that retains compatibility with the software tools developed for analyzing the results, takes a similar amount of time, and offers improved signal-to-noise ratio.
CRISPR knockout screening has mostly been performed by using viruses to deliver the required components into cells. In this paper, Xiong et al. demonstrate a virus-free approach that reduces noise and broadens access to CRISPR-based screens.
Chinese hamster ovary (CHO) cells are the most widely used mammalian host cells for the commercial production of therapeutic proteins. Fed-batch culture is widely used to produce therapeutic ...proteins, including monoclonal antibodies, because of its operational simplicity and high product titer. Despite technical advances in the development of culture media and cell cultures, it is still challenging to maintain high productivity in fed-batch cultures while also ensuring good product quality. In this review, factors that affect the quality attributes of therapeutic proteins in recombinant CHO (rCHO) cell culture, such as glycosylation, charge variation, aggregation, and degradation, are summarized and categorized into three groups: culture environments, chemical additives, and host cell proteins accumulated in culture supernatants. Understanding the factors that influence the therapeutic protein quality in rCHO cell culture will facilitate the development of large-scale, high-yield fed-batch culture processes for the production of high-quality therapeutic proteins.
•Fed-batch culture is widely used for commercial production of therapeutic proteins.•Ensuring good product quality in fed-batch cultures is still challenging.•Factors that affect product quality in recombinant CHO cell culture are summarized.•Understanding these factors is essential for the production of high-quality therapeutic proteins.
The ever-growing biopharmaceutical industry relies on the production of recombinant therapeutic proteins in Chinese hamster ovary (CHO) cells. The traditional timelines of CHO cell line development ...can be significantly shortened by the use of targeted gene integration (TI). However, broad use of TI has been limited due to the low specific productivity (q P) of TI-generated clones. Here, we show a 10-fold increase in the q P of therapeutic glycoproteins in CHO cells through the development and optimization of a multicopy TI method. We used a recombinase-mediated cassette exchange (RMCE) platform to investigate the effect of gene copy number, 5′ and 3′ gene regulatory elements, and landing pad features on q P. We evaluated the limitations of multicopy expression from a single genomic site as well as multiple genomic sites and found that a transcriptional bottleneck can appear with an increase in gene dosage. We created a dual-RMCE system for simultaneous multicopy TI in two genomic sites and generated isogenic high-producing clones with q P of 12–14 pg/cell/day and product titer close to 1 g/L in fed-batch. Our study provides an extensive characterization of the multicopy TI method and elucidates the relationship between gene copy number and protein expression in mammalian cells. Moreover, it demonstrates that TI-generated CHO cells are capable of producing therapeutic proteins at levels that can support their industrial manufacture.
Human cell lines are being increasingly used as host cells to produce therapeutic glycoproteins, due to their human glycosylation machinery. In an attempt to develop a platform for generating ...isogenic human cell lines producing therapeutic proteins based on targeted integration, three well-known human genomic safe harbors (GSHs)-AAVS1, CCR5, and human ROSA26 loci-were evaluated with respect to the transgene expression level and stability in human embryonic kidney (HEK293) cells. Among the three GSHs, the AAVS1 locus showed the highest eGFP expression with the highest homogeneity. Transgene expression at the AAVS1 locus was sustained without selection for approximately 3 months. Furthermore, the CMV promoter showed the highest expression, followed by the EF1α, SV40, and TK promoters at the AAVS1 locus. Master cell lines were created using CRISPR/Cas9-mediated integration of the landing pad into the AAVS1 locus and were used for faster generation of recombinant cell lines that produce therapeutic proteins with recombinase-mediated cassette exchange.
Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield ...protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO–K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.
•Genome-wide virus-free CRISPR/Cas9 screening was performed in CHO–K1 cells.•Novel and known hyperosmotic stress responsive gene clusters were identified.•Perturbation of identified genes conferred resistance to osmotic stress.•Knockout of Zfr and Pnp improved product titer under hyperosmotic conditions.
The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or ...glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine—the selection medium for Gs‐based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co‐transfection of Gs‐Enbrel and Aspg‐Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5‐fold higher specific productivity and ~3‐fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.
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