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Biomarkers are valuable tools in clinical research where they allow to predict susceptibility to diseases, or response to specific treatments. Likewise, biomarkers can be extremely useful in the biomanufacturing of therapeutic proteins. Indeed, constraints such as short timelines and the need to find hyper-productive cells could benefit from a data-driven approach during cell line and process development. Many companies still rely on large screening capacities to develop productive cell lines, but as they reach a limit of production, there is a need to go from empirical to rationale procedures. Similarly, during bioprocessing runs, substrate consumption and metabolism wastes are commonly monitored. None of them possess the ability to predict the culture behavior in the bioreactor. Big data driven approaches are being adapted to the study of industrial mammalian cell lines, enabled by the publication of Chinese hamster and CHO genome assemblies which allowed the use of next-generation sequencing with these cells, as well as continuous proteome and metabolome annotation. However, if these different -omics technologies contributed to the characterization of CHO cells, there is a significant effort remaining to apply this knowledge to biomanufacturing methods. The correlation of a complex phenotype such as high productivity or rapid growth to the presence or expression level of a specific biomarker could save time and effort in the screening of manufacturing cell lines or culture conditions. In this review we will first discuss the different biological molecules that can be identified and quantified in cells, their detection techniques, and associated challenges. We will then review how these markers are used during the different steps of cell line and bioprocess development, and the inherent limitations of this strategy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Improving current cell line development workflows can either focus on increasing the specific productivity of the cell lines or shortening timelines to reach the clinic as fast as possible. In this work, using the Beacon platform, we have combined two distinct protocols - early cloning with low-viability pools, and IgG membrane staining-, to concomitantly reach both objectives, and generate highly productive CHO clones in shorter timelines. Fast-sorting approaches using low-viability pools in combination with the Beacon platform have recently been reported to shorten CLD timelines. However, the low recovery led to a drastic reduction in the clone number obtained postcloning. Here, we report a combined approach of fast-sorting and fluorescent membrane staining. With this new protocol, the cells reach a correct recovery, allowing to fully exploit the Beacon screening capacities. In addition, by using a fluorescent staining recognizing the secreted IgG, we were able to enrich the fraction of highly secreting cells prior to cloning and we obtained significant increases in the cell's specific productivity. The combination of these two protocols has a synergistic effect, and as they help discarding the dead and nonproducing populations prior to cloning, they increase the throughput power of the Beacon platform and the detection of super productive clones., (© 2024 ARES Trading SA. Biotechnology Journal published by Wiley‐VCH GmbH.)

Mammalian cells have developed dedicated molecular mechanisms to tightly control expression levels of their genes where the specific transcriptomic signature across all genes eventually determines the cell's phenotype. Modulating cellular phenotypes is of major interest to study their role in disease or to reprogram cells for the manufacturing of recombinant products, such as biopharmaceuticals. Cells of mammalian origin, for example Chinese hamster ovary (CHO) and Human embryonic kidney 293 (HEK293) cells, are most commonly employed to produce therapeutic proteins. Early genetic engineering approaches to alter their phenotype have often been attempted by "uncontrolled" overexpression or knock-down/-out of specific genetic factors. Many studies in the past years, however, highlight that rationally regulating and fine-tuning the strength of overexpression or knock-down to an optimum level, can adjust phenotypic traits with much more precision than such "uncontrolled" approaches. To this end, synthetic biology tools have been generated that enable (fine-)tunable and/or inducible control of gene expression. In this review, we discuss various molecular tools used in mammalian cell lines and group them by their mode of action: transcriptional, post-transcriptional, translational and post-translational regulation. We discuss the advantages and disadvantages of using these tools for each cell regulatory layer and with respect to cell line engineering approaches. This review highlights the plethora of synthetic toolboxes that could be employed, alone or in combination, to optimize cellular systems and eventually gain enhanced control over the cellular phenotype to equip mammalian cell factories with the tools required for efficient production of emerging, more difficult-to-express biologics formats., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The integration of a transgene expression construct into the host genome is the initial step for the generation of recombinant cell lines used for biopharmaceutical production. The stability and level of recombinant gene expression in Chinese hamster ovary (CHO) can be correlated to the copy number, its integration site as well as the epigenetic context of the transgene vector. Also, undesired integration events, such as concatemers, truncated, and inverted vector repeats, are impacting the stability of recombinant cell lines. Thus, to characterize cell clones and to isolate the most promising candidates, it is crucial to obtain information on the site of integration, the structure of integrated sequence and the epigenetic status. Current sequencing techniques allow to gather this information separately but do not offer a comprehensive and simultaneous resolution. In this study, we present a fast and robust nanopore Cas9-targeted sequencing (nCats) pipeline to identify integration sites, the composition of the integrated sequence as well as its DNA methylation status in CHO cells that can be obtained simultaneously from the same sequencing run. A Cas9-enrichment step during library preparation enables targeted and directional nanopore sequencing with up to 724× median on-target coverage and up to 153 kb long reads. The data generated by nCats provides sensitive, detailed, and correct information on the transgene integration sites and the expression vector structure, which could only be partly produced by traditional Targeted Locus Amplification-seq data. Moreover, with nCats the DNA methylation status can be analyzed from the same raw data without prior DNA amplification., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Long non-coding RNAs (lncRNAs) are a potential new cell line engineering tool for improvement of yield and stability of CHO cells. In this study, we performed RNA sequencing of mAb producer CHO clones to study the lncRNA and protein coding transcriptome in relation to productivity. First, a robust linear model was used to identify genes correlating to productivity. To unravel specific patterns in expression of these genes, we employed weighted gene coexpression analysis (WGCNA) to find coexpressed modules, looking both for lncRNAs and coding genes. There was little overlap in the genes associated with productivity between the two products studied, possibly due to the difference in absolute range of productivity between the two mAbs. Therefore, we focused on the product with higher productivity and stronger candidate lncRNAs. To evaluate their potential as engineering targets, these candidate lncRNAs were transiently overexpressed or deleted by stable CRISPR Cas9 knock out both in a high and a low productivity subclone. We found that the thus achieved expression level of the identified lncRNAs, as confirmed by qPCR, does correlate well to productivity, so that they represent good markers that may be used for early clone selection. Additionally, we found that the deletion of one tested lncRNA region decreased viable cell density (VCD), prolonged culture time and increased cell size, final titer and specific productivity per cell. These results demonstrate the feasibility and usefulness of engineering lncRNA expression in production cell lines., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The glutamine synthetase (GS)-based Chinese hamster ovary (CHO) selection system is an attractive approach to efficiently identify suitable clones in the cell line generation process for biologics manufacture, for which GS-knockout (GS-KO) CHO cell lines are commonly used. Since genome analysis indicated that there are two GS genes in CHO cells, deleting only 1 GS gene could potentially result in the activation of other GS genes, consequently reducing the selection efficiency. Therefore, in this study, both GS genes identified on chromosome 5 (GS5) and 1 (GS1) of CHO-S and CHO-K1, were deleted using CRISPR/Cpf1. Both single and double GS-KO CHO-S and K1 showed robust glutamine-dependent growth. Next, the engineered CHO cells were tested for their efficiency of selection of stable producers of two therapeutic antibodies. Analysis of pool cultures and subclones after a single round of 25 µM methionine sulfoxinime (MSX) selection indicated that for CHO-K1 the double GS5,1-KO was more efficient as in the case of a single GS5-KO the GS1 gene was upregulated. In CHO-S, on the other hand, with an autologously lower level of expression of both variants of GS, a single GS5-KO was more robust and already enabled selection of high producers. In conclusion, CRISPR/Cpf1 can be efficiently used to knock out GS genes from CHO cells. The study also indicates that for the generation of host cell lines for efficient selection, the initial characterisation of expression levels of the target gene as well as the identification of potential escape mechanisms is important., (© 2023. The Author(s).)

Mammalian cells frequently encounter subtle perturbations during recombinant protein production. Identifying the genetic factors that govern the cellular stress response can facilitate targeted genetic engineering to obtain production cell lines that demonstrate a higher stress tolerance. To simulate nutrient stress, Chinese hamster ovary (CHO) cells were transferred into a glutamine(Q)-free medium and transcriptional dynamics using thiol(SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq) along with standard RNA-seq of stressed and unstressed cells were investigated. The SLAM-seq method allows differentiation between actively transcribed, nascent mRNA, and total (previously present) mRNA in the sample, adding an additional, time-resolved layer to classic RNA-sequencing. The cells tackle amino acid (AA) limitation by inducing the integrated stress response (ISR) signaling pathway, reflected in Atf4 overexpression in the early hours post Q deprivation, leading to subsequent activation of its targets, Asns, Atf3, Ddit3, Eif4ebp1, Gpt2, Herpud1, Slc7a1, Slc7a11, Slc38a2, Trib3, and Vegfa. The GCN2-eIF2α-ATF4 pathway is confirmed by a significant halt in transcription of translation-related genes at 24 h post Q deprivation. The downregulation of lipid synthesis indicates the inhibition of the mTOR pathway, further confirmed by overexpression of Sesn2. Furthermore, SLAM-seq detects short-lived transcription factors, such as Egr1, that would have been missed in standard experimental designs with RNA-seq. Our results describe the successful establishment of SLAM-seq in CHO cells and therefore facilitate its future use in other scenarios where dynamic transcriptome profiling in CHO cells is essential., (© 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Recombinant mammalian host cell lines, in particular CHO and HEK293 cells, are used for the industrial production of therapeutic proteins. Despite their well-known genomic instability, the control mechanisms that enable cells to respond to changes in the environmental conditions are not yet fully understood, nor do we have a good understanding of the factors that lead to phenotypic shifts in long-term cultures. A contributing factor could be inherent diversity in transcriptomes within a population. In this study, we used a full-length coverage single-cell RNA sequencing (scRNA-seq) approach to investigate and compare cell-to-cell variability and the impact of standardized and homogenous culture conditions on the diversity of individual cell transcriptomes, comparing suspension CHO-K1 and adherent HEK293FT cells. Our data showed a critical batch effect from the sequencing of four 96-well plates of CHO-K1 single cells stored for different periods of time, which was and may be therefore identified as a technical variable to consider in experimental planning. Besides, in an artificial and controlled culture environment such as used in routine cell culture technology, the gene expression pattern of a given population does not reveal any marker gene capable to disclose relevant cell population substructures, both for CHO-K1 cells and for HEK293FT cells. The variation observed is primarily driven by the cell cycle., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Giulia Borsi reports financial support was provided by University of Natural Resources and Life Sciences Vienna. Elly Sinkala, Max Sauerland and Julian Riba are employees of Cytena., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Modulation of expression levels of endogenous or recombinant genes can be of great interest for diverse applications, such as the study of genotype-phenotype relationships for a gene of interest, or fine-tuning of transcription to determine physiologically relevant effects of gene expression levels. During the last decades, several synthetic biology tools were established to control gene expression in mammalian cells such as Chinese hamster ovary (CHO) cells, one of the most important cell systems for basic research as well as the production of biopharmaceuticals. Here we describe the use of triplex forming oligos (TFOs), short RNA or ssDNA molecules that can bind to the major grove of their target duplex with great specificity, to control transgene expression in CHO cells. For proof of concept, a panel of TFOs with a size of 10-20 nts were designed with the help of the on-line tool Triplexator targeting the viral cytomegalovirus (CMV) promoter/enhancer region controlling the downstream reporter gene hCD4. The effect of TFOs was tested as ssDNA oligos pre-annealed to the promoter/enhancer region in vitro as well as upon endogenous transcription of the TFO as an RNA molecule binding to their target duplex in vivo. Results showed that not only binding of the TFO, but the exact location of triplex formation within the promoter/enhancer is paramount for transcription inhibition. After relieving a binding conflict by introducing a point mutation within the CMV promoter, longer TFOs (26-30 nts) could be designed and analysed. Selected TFOs achieved a reduction in recombinant hCD4 expression of up to 85% in CHO-K1 cells., Competing Interests: Declaration of Competing Interest The authors declare no commercial or financial conflict of interest. Sybille Galosy is a current GSK employee and holds stock options., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Competing Interests: Declaration of competing interest We have no conflict of interest.

Nafamostat is an approved short-acting serine protease inhibitor. However, its administration is also associated with anaphylactic reactions. One mechanism to augment hypersensitivity reactions could be inhibition of diamine oxidase (DAO). The chemical structure of nafamostat is related to the potent DAO inhibitors pentamidine and diminazene. Therefore, we tested whether nafamostat is a human DAO inhibitor. Using different activity assays, nafamostat reversibly inhibited recombinant human DAO with an IC 50 of 300-400 nM using 200 µ M substrate concentrations. The K i of nafamostat for the inhibition of putrescine and histamine deamination is 27 nM and 138 nM, respectively For both substrates, nafamostat is a mixed mode inhibitor with P values of <0.01 compared with other inhibition types. Using 80-90% EDTA plasma, the IC 50 of nafamostat inhibition was approximately 360 nM using 20 µ M cadaverine. In 90% EDTA plasma, the IC 50 concentrations were 2-3 µ M using 0.9 µ M and 0.18 µ M histamine as substrate. In silico modeling showed a high overlap compared with published diminazene crystallography data, with a preferred orientation of the guanidine group toward topaquinone. In conclusion, nafamostat is a potent human DAO inhibitor and might increase severity of anaphylactic reaction by interfering with DAO-mediated extracellular histamine degradation. SIGNIFICANCE STATEMENT: Treatment with the short-acting anticoagulant nafamostat during hemodialysis, leukocytapheresis, extracorporeal membrane oxygenator procedures, and disseminated intravascular coagulation is associated with severe anaphylaxis in humans. Histamine is a central mediator in anaphylaxis. Potent inhibition of the only extracellularly histamine-degrading enzyme diamine oxidase could augment anaphylaxis reactions during nafamostat treatment., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Recent advances in omics technologies and the broad availability of big datasets have revolutionized our understanding of Chinese hamster ovary cells in their role as the most prevalent host for production of complex biopharmaceuticals. In consequence, our perception of this "workhorse of the biopharmaceutical industry" has successively shifted from that of a nicely working, but unknown recombinant protein producing black box to a biological system governed by multiple complex regulatory layers that might possibly be harnessed and manipulated at will. Despite the tremendous progress that has been made to characterize CHO cells on various omics levels, our understanding is still far from complete. The well-known inherent genetic plasticity of any immortalized and rapidly dividing cell line also characterizes CHO cells and can lead to problematic instability of recombinant protein production. While the high mutational frequency has been a focus of CHO cell research for decades, the impact of epigenetics and its role in differential gene expression has only recently been addressed. In this review we provide an overview about the current understanding of epigenetic regulation in CHO cells and discuss its significance for shaping the cell's phenotype. We also look into current state-of-the-art technology that can be applied to harness and manipulate the epigenetic network so as to nudge CHO cells towards a specific phenotype. Here, we revise current strategies on site-directed integration and random as well as targeted epigenome modifications. Finally, we address open questions that need to be investigated to exploit the full repertoire of fine-tuned control of multiplexed gene expression using epigenetic and systems biology tools., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Elevated plasma and tissues histamine concentrations can cause severe symptoms in mast cell activation syndrome, mastocytosis or anaphylaxis. Endogenous and recombinant human diamine oxidase (rhDAO) can rapidly and completely degrade histamine, and administration of rhDAO represents a promising new treatment approach for diseases with excess histamine release from activated mast cells. We recently generated heparin-binding motif mutants of rhDAO with considerably increased in vivo half-lives in rodents compared with the rapidly cleared wildtype protein. Herein, we characterize the role of an evolutionary recently added glycosylation site asparagine 168 in the in vivo clearance and the influence of an unusually solvent accessible free cysteine 123 on the oligomerization of diamine oxidase (DAO). Mutation of the unpaired cysteine 123 strongly reduced oligomerization without influence on enzymatic DAO activity and in vivo clearance. Recombinant hDAO produced in ExpiCHO-S™ cells showed a 15-fold reduction in the percentage of glycans with terminal sialic acid at Asn168 compared with Chinese hamster ovary (CHO)-K1 cells. Capping with sialic acid was also strongly reduced at the other glycosylation sites. The high abundance of terminal mannose and N-acetylglucosamine residues in the four glycans expressed in ExpiCHO-S™ cells compared with CHO-K1 cells resulted in rapid in vivo clearance. Mutation of Asn168 or sialidase treatment also significantly increased clearance. Intact N-glycans at Asn168 seem to protect DAO from rapid clearance in rodents. Full processing of all glycoforms is critical for preserving the improved in vivo half-life characteristics of the rhDAO heparin-binding motif mutants., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To evaluate the contribution of endogenous diamine oxidase (DAO) in the inactivation of exogenous histamine, to find a mouse strain with increased histamine sensitivity and to test the efficacy of rhDAO in a histamine challenge model., Methods: Diamine oxidase knockout (KO) mice were challenged with orally and subcutaneously administered histamine in combination with the β-adrenergic blocker propranolol, with the two histamine-N-methyltransferase (HNMT) inhibitors metoprine and tacrine, with folic acid to mimic acute kidney injury and treated with recombinant human DAO. Core body temperature was measured using a subcutaneously implanted microchip and histamine plasma levels were quantified using a homogeneous time resolved fluorescence assay., Results: Core body temperature and plasma histamine levels were not significantly different between wild type (WT) and DAO KO mice after oral and subcutaneous histamine challenge with and without acute kidney injury or administration of HNMT inhibitors. Treatment with recombinant human DAO reduced the mean area under the curve (AUC) for core body temperature loss by 63% (p = 0.002) and the clinical score by 88% (p < 0.001). The AUC of the histamine concentration was reduced by 81%., Conclusions: Inactivation of exogenous histamine is not driven by enzymatic degradation and kidney filtration. Treatment with recombinant human DAO strongly reduced histamine-induced core body temperature loss, histamine concentrations and prevented the development of severe clinical symptoms., (© 2022. The Author(s).)

Metabolic modelling strives to develop modelling approaches that are robust and highly predictive. To achieve this, various modelling designs, including hybrid models, and parameter estimation methods that define the type and number of parameters used in the model, are adapted. Accurate input data play an important role so that the selection of experimental methods that provide input data of the required precision with low measurement errors is crucial. For the biopharmaceutically relevant protein glycosylation, the most prominent available models are kinetic models which are able to capture the dynamic nature of protein N-glycosylation. In this review we focus on how to choose the most suitable model for a specific research question, as well as on parameters and considerations to take into account before planning relevant experiments., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Excessive plasma histamine concentrations cause symptoms in mast cell activation syndrome, mastocytosis, or anaphylaxis. Anti-histamines are often insufficiently efficacious. Human diamine oxidase (hDAO) can rapidly degrade histamine and therefore represents a promising new treatment strategy for conditions with pathological histamine concentrations., Methods: Positively charged amino acids of the heparin-binding motif of hDAO were replaced with polar serine or threonine residues. Binding to heparin and heparan sulfate, cellular internalization and clearance in rodents were examined., Results: Recombinant hDAO is rapidly cleared from the circulation in rats and mice. After mutation of the heparin-binding motif, binding to heparin and heparan sulfate was strongly reduced. The double mutant rhDAO-R568S/R571T showed minimal cellular uptake. The short α-distribution half-life of the wildtype protein was eliminated, and the clearance was significantly reduced in rodents., Conclusions: The successful decrease in plasma clearance of rhDAO by mutations of the heparin-binding motif with unchanged histamine-degrading activity represents the first step towards the development of rhDAO as a first-in-class biopharmaceutical to effectively treat diseases characterized by excessive histamine concentrations in plasma and tissues., Funding: Austrian Science Fund (FWF) Hertha Firnberg program grant T1135 (EG); Sigrid Juselius Foundation, Medicinska Understödsförening Liv och Hälsa rft (TAS and SeV)., Competing Interests: EG, KT, KP, MS, SV, SV, GK, JP, TS, NB, TB is named as an inventor with The Medical University of Vienna and the University of Natural Resources and Life Sciences of a patent describing the rhDAO heparin-binding motif mutants presented herein (patent pending WO2020169577A1), KS none, MR None, BJ is named as an inventor with The Medical University of Vienna and the University of Natural Resources and Life Sciences of a patent describing the rhDAO heparin-binding motif mutants presented herein (patent pending WO2020169577A1)., (© 2021, Gludovacz et al.)

With the emergence of new CRISPR/dCas9 tools that enable site specific modulation of DNA methylation and histone modifications, more detailed investigations of the contribution of epigenetic regulation to the precise phenotype of cells in culture, including recombinant production subclones, is now possible. These also allow a wide range of applications in metabolic engineering once the impact of such epigenetic modifications on the chromatin state is available. In this study, enhanced DNA methylation tools were targeted to a recombinant viral promoter (CMV), an endogenous promoter that is silenced in its native state in CHO cells, but had been reactivated previously (β-galactoside α-2,6-sialyltransferase 1) and an active endogenous promoter (α-1,6-fucosyltransferase), respectively. Comparative ChIP-analysis of histone modifications revealed a general loss of active promoter histone marks and the acquisition of distinct repressive heterochromatin marks after targeted methylation. On the other hand, targeted demethylation resulted in autologous acquisition of active promoter histone marks and loss of repressive heterochromatin marks. These data suggest that DNA methylation directs the removal or deposition of specific histone marks associated with either active, poised or silenced chromatin. Moreover, we show that de novo methylation of the CMV promoter results in reduced transgene expression in CHO cells. Although targeted DNA methylation is not efficient, the transgene is repressed, thus offering an explanation for seemingly conflicting reports about the source of CMV promoter instability in CHO cells. Importantly, modulation of epigenetic marks enables to nudge the cell into a specific gene expression pattern or phenotype, which is stabilized in the cell by autologous addition of further epigenetic marks. Such engineering strategies have the added advantage of being reversible and potentially tunable to not only turn on or off a targeted gene, but also to achieve the setting of a desirable expression level., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism., (Copyright © 2021 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)