Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening.

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.

Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium.

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.

Accelerated homology-directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and fluorescent enrichment.

Targeted gene integration into site-specific loci can be achieved in Chinese hamster ovary (CHO) cells via CRISPR/Cas9 genome editing technology and the homology-directed repair (HDR) pathway. The low efficiency of HDR often requires antibiotic selection, which limits targeted integration of multiple genes at multiple sites. To improve HDR-mediated targeted integration, while avoiding the use of selection markers, chemical treatment for increased HDR, and fluorescent enrichment of genome-edited cells was assessed in CHO cells. Chemical treatment did not improve HDR-mediated targeted integration. In contrast, fluorescent markers in Cas9 and donor constructs enable FACS enrichment, resulting in a threefold increase in the number of cells with HDR-mediated genome editing. Combined with this enrichment method, large transgenes encoding model proteins (including an antibody) were successfully targeted integrated. This approach provides a simple and fast strategy for targeted generation of stable CHO production cell lines in a rational way. Biotechnol. Bioeng. 2016;113: 2518-2523. © 2016 Wiley Periodicals, Inc.

Characterisation of two snake toxin-targeting human monoclonal immunoglobulin G antibodies expressed in tobacco plants.

Current snakebite antivenoms are based on polyclonal animal-derived antibodies, which can neutralize snake venom toxins in envenomed victims, but which are also associated with adverse reactions. Therefore, several efforts within antivenom research aim to explore the utility of recombinant monoclonal antibodies, such as human immunoglobulin G (IgG) antibodies, which are routinely used in the clinic for other indications. In this study, the feasibility of using tobacco plants as bioreactors for expressing full-length human monoclonal IgG antibodies against snake toxins was investigated. We show that the plant-produced antibodies perform similarly to their mammalian cell-expressed equivalents in terms of in vitro antigen binding. Complete neutralization was achieved by both the plant and mammalian cell-produced anti-α-cobratoxin antibody. The feasibility of using plant-based expression systems may potentially make it easier for laboratories in resource-poor settings to work with human monoclonal IgG antibodies.

Assessing developability early in the discovery process for novel biologics.

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.

Factors affecting the quality of therapeutic proteins in recombinant Chinese hamster ovary cell culture.

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.

An optimized genome-wide, virus-free CRISPR screen for mammalian cells.

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.

Erratum: An optimized genome-wide, virus-free CRISPR screen for mammalian cells.

[This corrects the article DOI: 10.1016/j.crmeth.2021.100062.].

Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells.

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 (qP) of TI-generated clones. Here, we show a 10-fold increase in the qP 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 qP. 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 qP 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.

Comprehensive Analysis of Genomic Safe Harbors as Target Sites for Stable Expression of the Heterologous Gene in HEK293 Cells.

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.

BCAT1 and BCAT2 disruption in CHO cells has cell line-dependent effects.

In recombinant protein expression using Chinese hamster ovary (CHO) cells, chemically defined media contain essential amino acids such as branched chain amino acids (BCAAs) leucine, isoleucine and valine. Availability of amino acids is critical as these are building blocks for protein synthesis. However, breakdown of amino acids can lead to build up of toxic intermediates and metabolites that decrease cell growth, productivity and product quality. BCAA catabolism also hampers the usage of BCAAs for protein synthesis. In this work we studied the effects of disrupting the genes responsible for the first step of BCAA catabolism: branched chain aminotransferase 1 (Bcat1) and branched chain aminotransferase 2 (Bcat2). We evaluated the effect of disrupting the genes individually and in combination, and examined the effects in producer and non-producer host cells. Our experiments show that Bcat1 disruption improves cell growth in producer cells, but not in non-producers. Conversely, Bcat2 has a minor negative effect on growth in producer cells, and none in non-producers. Combined Bcat1 and Bcat2 disruption improves growth in producer cells. By-product metabolism is cell line-, clone- and producer-dependent. Overall, our results show that the effects of targeting Bcat1 and/or Bcat2 are cell line-dependent, and seemingly linked to the burden of recombinant protein expression.

Systematic Evaluation of Site-Specific Recombinant Gene Expression for Programmable Mammalian Cell Engineering.

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.

Minimizing Clonal Variation during Mammalian Cell Line Engineering for Improved Systems Biology Data Generation.

Mammalian cells are widely used to express genes for basic biology studies and biopharmaceuticals. Current methods for generation of engineered cell lines introduce high genomic and phenotypic diversity, which hamper studies of gene functions and discovery of novel cellular mechanisms. Here, we minimized clonal variation by integrating a landing pad for recombinase-mediated cassette exchange site-specifically into the genome of CHO cells using CRISPR and generated subclones expressing four different recombinant proteins. The subclones showed low clonal variation with high consistency in growth, transgene transcript levels and global transcriptional response to recombinant protein expression, enabling improved studies of the impact of transgenes on the host transcriptome. Little variation over time in subclone phenotypes and transcriptomes was observed when controlling environmental culture conditions. The platform enables robust comparative studies of genome engineered CHO cell lines and can be applied to other mammalian cells for diverse biological, biomedical and biotechnological applications.

Application of CRISPR/Cas9 Genome Editing to Improve Recombinant Protein Production in CHO Cells.

Genome editing has become an increasingly important aspect of Chinese Hamster Ovary (CHO ) cell line engineering for improving production of recombinant protein therapeutics. Currently, the focus is directed toward expanding the product diversity, controlling and improving product quality and yields. In this chapter, we present our protocol on how to use the genome editing tool Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) to knockout engineering target genes in CHO cells. As an example, we refer to the glutamine synthetase (GS)-encoding gene as the knockout target gene, a knockout that increases the selection efficiency of the GS-mediated gene amplification system.

CRISPR/Cas9-mediated genome engineering of CHO cell factories: Application and perspectives.

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.

One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment.

The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9-expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off-target effects were observed from off-target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene-disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild-type CHO-S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further.

Versatile microscale screening platform for improving recombinant protein productivity in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are widely used as cell factories for the production of biopharmaceuticals. In contrast to the highly optimized production processes for monoclonal antibody (mAb)-based biopharmaceuticals, improving productivity of non-mAb therapeutic glycoproteins is more likely to reduce production costs significantly. The aim of this study was to establish a versatile target gene screening platform for improving productivity for primarily non-mAb glycoproteins with complete interchangeability of model proteins and target genes using transient expression. The platform consists of four techniques compatible with 96-well microplates: lipid-based transient transfection, cell cultivation in microplates, cell counting and antibody-independent product titer determination based on split-GFP complementation. We were able to demonstrate growth profiles and volumetric productivity of CHO cells in 96-half-deepwell microplates comparable with those obtained in shake flasks. In addition, we demonstrate that split-GFP complementation can be used to accurately measure relative titers of therapeutic glycoproteins. Using this platform, we were able to detect target gene-specific increase in titer and specific productivity of two non-mAb glycoproteins. In conclusion, the platform provides a novel miniaturized and parallelisable solution for screening target genes and holds the potential to unravel genes that can enhance the secretory capacity of CHO cells.