Although PM2.5 toxicity is known to be related to its chemical composition, the effect of interactions among various particles' components on the toxicity is not well explored. To understand these ...interactions, especially metals and organic compounds on PM2.5 cytotoxicity, we chose several redox-active substances known to be present in the ambient particles such as metals (Cu, Fe, and Mn) and quinones 9,10-phenanthraquinone (PQ), 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ), and 5-hydroxy-1,4-naphthoquinone (5,H-1,4-NQ). Cytotoxicity was assessed through a Chinese hamster ovary (CHO) cells assay and expressed by a median lethal concentration (LC50). Two methods were employed to assess the interactions. In the first method, we tested the impact of nontoxic level of a component on the LC50 of other components. In the second method, we mixed two components in different concentration ratios to expose the cells and calculated a mixture toxicity index (MTI). MTI is a composite value to quantify the nature of interactions such that the interactions are considered synergistic when MTI > 1, additive when 0 < MTI ≤ 1 and antagonistic when MTI < 0. The interactions between quinones and metals were largely synergistic by both methods. To further assess the environmental relevance of these mixtures, we extracted organic compounds termed as water-soluble Humic-like substances (HULIS) from real ambient PM samples and mixed them with individual metals. A similar pattern, as observed from the interaction of quinones and metals, was found. Moreover, the interactions became more synergistic as the relative concentration of metals with respect to water-soluble HULIS was decreased in these mixtures. With environmentally relevant mass concentration ratios of organics to metals (75–7500), the interactions were strongly synergistic (MTI = 1–115). These results indicate the importance of incorporating the interaction among various PM components for estimating the net toxicity of ambient PM2.5.
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•We systematically explored the effect of interaction among various PM2.5 components on the cytotoxicity.•The interactions between metals and organic compounds for the cytotoxicity appear to be concentration-dependent.•Under the environmentally-relevant concentration ratios, the interactions of metals and organic compounds are synergistic.•To estimate the overall toxicity of PM2.5, the interaction effects among different PM components should be considered.
The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of ...biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality. With the growing toolbox of genetic engineering technologies, it is now possible to address general as well as host‐ or biopharmaceutical‐specific product quality obstacles.
In this review, we present diverse expression systems derived from mammalians, bacteria, yeast, plants, and insects as well as available genetic engineering tools. We focus on genes for knockout/knockdown and overexpression for meaningful approaches to improve biopharmaceutical PTMs and discuss their applicability as well as future trends in the field.
The number of approved biopharmaceuticals, where product quality attributes remain of major importance, is increasing steadily. Within the available variety of expression hosts, the production of biopharmaceuticals faces diverse limitations with respect to posttranslational modifications (PTM), while different biopharmaceuticals demand different forms and specifications of PTMs for proper functionality.
•Comparison of batch, continuous and hybrid modes in monoclonal antibody production.•A modified kinetic model for the main cultivation unit.•Model validation with experimental data from literature ...and a pilot facility.•Analysis of production cost and time for individual units and integrated scenarios.•Analysis for production scales from clinical to commercial with single-use equipment.
Monoclonal antibodies are leading the growing biopharmaceutical markets. To sustain the increasing demand, manufacturing processes should be optimized to increase efficiency and flexibility. This work investigates the impacts of the application of single-use equipment in batch, continuous, and hybrid scenarios. Operating costs and time are compared at different production scales from clinical to commercial manufacturing for individual unit operations and for the final integrated scenarios. A modified kinetic model is presented for the main cultivation unit. The model is validated using experimental data from literature and from a pilot facility using Chinese hamster ovary cells. Integrated scenarios with fed-batch cultivation and continuous capture have the lowest operating costs. The selection of operating modes across production scales is discussed along with further considerations for the design of integrated processes. The presented models could be used for the development of process design, control, and scheduling efforts for achieving sustainable biopharmaceutical manufacturing.
•UCOE element was associated with increased yield of antibody.•UCOE pools had greater number of positive clones and more stable expression.•Clones that stably co-express mAb and its specific SINEUP ...had more mAb titer.•Stably expressed SINEUP clones had stable long-term expression.•Combined UCOE and SINEUP provided more optimized expression conditions in CHO cells.
The growing need for monoclonal antibodies (mAbs) necessitates the development of novel and efficient production approaches. Regulatory elements like ubiquitous chromatin-opening elements (UCOEs) have been employed for improvement of the mAb expression in the Chinese hamster ovary (CHO) cells. SINEUPs are a class of long non-coding RNAs, which can improve the translation of partly overlapping mRNAs. A combination of these two elements might lead to higher production of mAbs. Therefore, the current study was conducted to investigate the effects of SINEUPs and A2UCOE on the expression of an IgG1 in the CHO-K1 cells. Hence, after constructing the mAb, mAb-SINEUP, and mAb-UCOE vectors, four stable cell pools were generated through combining the above vectors. According to the expression analysis, antibody yields were higher in the mAb-SINEUP and mAb-UCOE cell pools compared to the mAb cells. In addition, the cells possessing both SINEUP and UCOE elements provided the best expression. Persistent mAb expression was observed for over 2 months in these cells, whilst the expression was decreased in the mAb pool. SINEUP and UCOE positively influenced the stable mAb expression. It can be concluded that the SINEUP and UCOE enhance the antibody stability and expression level separately and their combination improves the mAb production in the CHO cells.
In recent years, acceleration of development timelines has become a major focus within the biopharmaceutical industry to bring innovative therapies faster to patients. However, in order to address a ...high unmet medical need even faster further acceleration potential has to be identified to transform “speed-to-clinic” concepts into “warp-speed” development programs. Recombinant Chinese hamster ovary (CHO) cell lines are the predominant expression system for monoclonal antibodies (mAbs) and are routinely generated by random transgene integration (RTI) of the genetic information into the host cell genome. This process, however, exhibits considerable challenges such as the requirement for a time-consuming clone screening process to identify a suitable clonally derived manufacturing cell line. Hence, RTI represents an error prone and tedious method leading to long development timelines until availability of Good Manufacturing Practice (GMP)-grade drug substance (DS). Transposase-mediated semi-targeted transgene integration (STI) has been recently identified as a promising alternative to RTI as it allows for a more rapid generation of high-performing and stable production cell lines. In this report, we demonstrate how a STI technology was leveraged to develop a very robust DS manufacturing process based on a stable pool cell line at unprecedented pace. Application of the novel strategy resulted in the manufacturing of GMP-grade DS at 2,000 L scale in less than three months paving the way for a start of Phase I clinical trials only six months after transfection. Finally, using a clonally derived production cell line, which was established from the parental stable pool, we were able to successfully implement a process with an increased mAb titer of up to 5 g per liter at the envisioned commercial scale (12,000 L) within eight months.
•Chinese hamster ovary cells.•Cell line development.•Transposase.•Acceleration.•Monoclonal antibody.
High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this ...results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties.
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The metabolism of Chinese Hamster Ovary (CHO) cells in a production environment has been extensively investigated. However, a key metabolic transition, the switch from lactate production to lactate ...consumption, remains enigmatic. Though commonly observed in CHO cultures, the mechanism(s) by which this metabolic shift is triggered is unknown. Despite this, efforts to control the switch have emerged due to the association of lactate consumption with improved cell growth and productivity. This review aims to consolidate current theories surrounding the lactate switch. The influence of pH, NAD+/NADH, pyruvate availability and mitochondrial function on lactate consumption are explored. A hypothesis based on the cellular redox state is put forward to explain the onset of lactate consumption. Various techniques implemented to control the lactate switch, including manipulation of the culture environment, genetic engineering, and cell line selection are also discussed.
A switch in lactate metabolism is frequently observed in a CHO cell production environment. Cultures transition from a lactate producing to a lactate consuming state, a feature which is associated with enhanced process performance. However, the molecular mechanism through which this switch is conferred remains enigmatic. This review uses existing data to argue that redox imbalance is the primary driver of this metabolic switch.
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.
Recent studies have unveiled the unique roles of extracellular vesicles (EVs) in various cellular processes including protein degradation, transport, and intercellular communication. However, the EVs ...of Chinese hamster ovary (CHO) cells, the workhorse of biologics manufacturing, have not been well-characterized despite their significant roles in protein production. Herein, we successfully isolated CHO EVs from CHO fed-batch cultures and identified their messenger RNA (mRNA) and micro RNA (miRNA) contents through next-generation sequencing. We found that mRNAs corresponding to oxidative phosphorylation were highly enriched in microvesicles (large EVs) but absent in exosomes (small EVs). We also found that both large EVs and small EVs had enriched mRNA species corresponding to key signaling pathways for cell proliferation, survival, and growth, including the TGFβ and PI3K/Akt pathways. In addition, the enrichment of miR-196a-5p in both small EVs and large EVs suggests an anti-apoptotic and proliferative function for EVs through intercellular communication. The identification of these mRNAs and miRNAs associated with cell growth and survival sheds light on the potential role of extracellular vesicles in the context of biologics manufacturing and may help further optimize CHO biologics production.
•RNA content (mRNAs and miRNAs) of Extracellular Vehicles (EVs) from Chinese hamster ovary (CHO) cells has been sequenced.•mRNAs corresponding to oxidative phosphorylation were highly enriched in large EVs but absent in small EVs.•CHO EVs had enriched mRNA species corresponding to key signaling pathways for cell proliferation, survival, and growth.•miR-196a-5p was enriched in CHO EVs suggesting an anti-apoptotic and proliferative function.
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