In eukaryotes, the localization of small ribosomal subunits to mRNA transcripts requires the translation of Kozak elements at the starting site. The sequence of Kozak elements affects the translation ...efficiency of protein synthesis. However, whether the upstream nucleotide of Kozak sequence affects the expression of recombinant proteins in Chinese hamster ovary (CHO) cells remains unclear. In order to find the optimal sequence to enhance recombinant proteins expression in CHO cells, −10 to +4 sequences around ATG in 100 CHO genes were compared, and the extended Kozak elements with different translation intensities were constructed. Using the classic Kozak element as control, the effects of optimized extended Kozak elements on the secreted alkaline phosphatase (SEAP) and human serum albumin (HSA) gene were studied. The results showed that the optimized extended Kozak sequence can enhance the stable expression level of recombinant proteins in CHO cells. Furthermore, it was found that the increased expression level of the recombinant protein was not related with higher transcription level. In summary, optimizing extended Kozak elements can enhance the expression of recombinant proteins in CHO cells, which contributes to the construction of an efficient expression system for CHO cells.
•Kozak sequences were optimized based on bioinformatics analysis.•Optimization of Kozak can increase recombinant protein expression in CHO cell.•Kozak element increased recombinant protein expression was not caused by transcription level enhancing.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The reliability and methodology of genome-scale metabolic models (GEMs) of Chinese hamster ovary (CHO) cells have advanced.CHO-GEMs have aided in cell line and process development, thus impacting on ...biomanufacturing efficiency.An integrative model structure can incorporate multiple layers and capture condition-specific cell regulation.Integration of CHO-GEMs with artificial intelligence (AI) and advanced algorithms will enable autonomous bioreactor management for digital biomanufacturing.
Genome-scale metabolic models (GEMs) of Chinese hamster ovary (CHO) cells are valuable for gaining mechanistic understanding of mammalian cell metabolism and cultures. We provide a comprehensive overview of past and present developments of CHO-GEMs and in silico methods within the flux balance analysis (FBA) framework, focusing on their practical utility in rational cell line development and bioprocess improvements. There are many opportunities for further augmenting the model coverage and establishing integrative models that account for different cellular processes and data for future applications. With supportive collaborative efforts by the research community, we envisage that CHO-GEMs will be crucial for the increasingly digitized and dynamically controlled bioprocessing pipelines, especially because they can be successfully deployed in conjunction with artificial intelligence (AI) and systems engineering algorithms.
Genome-scale metabolic models (GEMs) of Chinese hamster ovary (CHO) cells are valuable for gaining mechanistic understanding of mammalian cell metabolism and cultures. We provide a comprehensive overview of past and present developments of CHO-GEMs and in silico methods within the flux balance analysis (FBA) framework, focusing on their practical utility in rational cell line development and bioprocess improvements. There are many opportunities for further augmenting the model coverage and establishing integrative models that account for different cellular processes and data for future applications. With supportive collaborative efforts by the research community, we envisage that CHO-GEMs will be crucial for the increasingly digitized and dynamically controlled bioprocessing pipelines, especially because they can be successfully deployed in conjunction with artificial intelligence (AI) and systems engineering algorithms.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
For over three decades, Chinese hamster ovary (CHO) cells have been the chosen expression platform for the production of therapeutic proteins with complex post‐translational modifications. However, ...the metabolism of these cells is far from perfect and optimized, and requires substantial know how and process optimization and monitoring to perform efficiently. One of the main reasons for this is the production and accumulation of toxic and growth‐inhibiting metabolites during culture. Lactate and ammonium are the most known, but many more have been identified. In this review, an overview of metabolites that deplete and accumulate throughout the course of cultivations with toxic and growth inhibitory effects to the cells is presented. Further, an overview of the CHO metabolism with emphasis to metabolic pathways of amino acids, glutathione (GSH), and related compounds which have growth‐inhibiting and/or toxic effect on the cells is provided. Additionally, relevant publications which describe the applications of metabolomics as a powerful tool for revealing which reactions occur in the cell under certain conditions are surveyed and growth‐inhibiting and toxic metabolites are identified. Also, a number of resources that describe the cellular mechanisms of CHO and are available on‐line are presented. Finally, the application of this knowledge for bioprocess and medium development and cell line engineering is discussed.
Chinese hamster ovary (CHO) cells are the chosen expression platform for the production of therapeutic proteins. ‘Omics data sets can aid and guide the rational design and generation of mammalian cells factories free of unwanted metabolic products and therefore capable of achieving higher cell densities and productivities. In this review, a number of metabolites depleting and accumulating throughout the course of cultivations, that have toxic and growth inhibitory effects to the cells are presented. Additionally, the potential application of this knowledge along with the use of analytical methods and on‐line metabolic resources for enhancing CHO cell lines by combining media development and cell line engineering approaches is discussed.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Microfiltration membranes are increasingly used to retain the cells inside bioreactors while continuous harvest of the desired biopharmaceutical occurs in perfusion processes. One method of ...microfiltration is Alternating Tangential Flow (ATF) filtration, which involves moving the cultivation broth tangentially back and forth across the membrane, which results in a backwash effect that reduces fouling. In this study, fouling was investigated with asymmetric polysulfone hollow fibers operated in ATF mode attached to a bioreactor producing a recombinant protein in Chinese Hamster Ovary (CHO) cells. Fouling was assessed through different approaches, including determination of critical flux using an improved flux-step method. Fouling was studied through measurements of Transmembrane Pressure (TMP), protein transmission, membrane pore size and staining of the membrane after operation to visualize the distribution of biological fouling inside the membrane. For critical flux determination, fluxes of up to 69 LMH were used without exceeding the critical flux. No sign of fouling was observed for the short-term (<3.5 h) critical flux experiment. However, during prolonged operation at 8.3 LMH the TMP jumped to 0.9–0.95 bar, indicating fouling. At this state, the protein transmission remains at the same high level (>88 %).
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•Mammalian cell culturing for production of recombinant protein.•Reverse asymmetric membrane hollow fiber filtration in perfusion process.•Critical flux determination in Alternating Tangential Flow Filtration.•Distribution of biological fouling in membrane by staining techniques.•Constantly high protein transmission even though significant fouling is present.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
5.
Heparin: Past, Present, and Future Oduah, Eziafa I; Linhardt, Robert J; Sharfstein, Susan T
Pharmaceuticals (Basel, Switzerland),
07/2016, Volume:
9, Issue:
3
Journal Article
Peer reviewed
Open access
Heparin, the most widely used anticoagulant drug in the world today, remains an animal-derived product with the attendant risks of adulteration and contamination. A contamination crisis in 2007-2008 ...increased the impetus to provide non-animal-derived sources of heparin, produced under cGMP conditions. In addition, recent studies suggest that heparin may have significant antineoplastic activity, separate and distinct from its anticoagulant activity, while other studies indicate a role for heparin in treating inflammation, infertility, and infectious disease. A variety of strategies have been proposed to produce a bioengineered heparin. In this review, we discuss several of these strategies including microbial production, mammalian cell production, and chemoenzymatic modification. We also propose strategies for creating "designer" heparins and heparan-sulfates with various biochemical and physiological properties.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Perfusion cell cultures generate higher viable cell densities (VCD) and volumetric productivity compared to fed-batch cultures. However, due to the limited availability of small-scale perfusion ...models, perfusion systems are seldom used to produce licensed biotherapeutics. This study evaluated two small-scale perfusion mimic protocols to bridge the research-to-production gap. Shake flasks and the ambr250 HT were used to compare centrifugation and in situ gravity settling protocols. The centrifugation protocol achieved a peak VCD >50 million cells/mL and is well-suited to media formulation and feeding strategy comparisons. The in situ gravity settling protocol achieved ∼20 million cells/mL and is well-suited to cell productivity and stability studies. The cell retention steps resulted in temporary DO and pH changes but did not affect the overall culture's health. Further, both protocols were able to recover from an imposed long-duration DO stress, although the centrifugation protocol cultures had higher cell specific oxygen consumption rates indicative of higher culture stress. Both protocols sustained the cultures for 39 days with stable cell specific productivities (∼27 pg/cell·day). Overall, this study demonstrated the feasibility of two economic small-scale perfusion mimic protocols in a standard small-scale bioreactor system, which could be translated to other multi-unit small-scale bioreactor systems.
•Centrifugation perfusion mimic protocol enables media formulation comparisons.•Centrifugation perfusion mimic protocol assists feeding strategy comparisons.•In situ gravity perfusion mimic protocols evaluates cell productivity stability.•Cell specific oxygen uptake rates indicate increased stress for CHO cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Monoclonal antibodies (mAbs) represent a significant segment of biopharmaceuticals, with the market for mAb therapeutics expected to reach $200 billion in 2021. Chinese Hamster Ovary (CHO) cells are ...the industry standard for large-scale mAb production owing to their adaptability and genetic engineering capabilities. However, maintaining consistent product quality is challenging, primarily because of the inherent genetic instability of CHO cells. In this study, we address the need for advanced technologies for quality monitoring of host cells in biopharmaceuticals. We highlight the limitations of traditional cell assessment techniques such as flow cytometry and propose a noninvasive, label-free image-based analysis method. By utilizing advanced image processing and machine learning, this technique aims to non-invasively and quantitatively evaluate subtle quality changes in suspension cells. The research aims to investigate the use of morphological analysis for identifying subtle alterations in mAb productivity of CHO cells, employing cells stimulated by compounds as a model for this study. Our results show that the mAb productivity of CHO cells (day 8) can be predicted only from their early morphological profile (day 3). Our study also discusses the importance of strategic methods for forecasting host cell mAb productivity using morphological profiles, as inferred from our machine learning models specialized in predictive score prediction and anomaly prediction.
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•Stable image-based analysis for suspension cells was established.•Early morphological profile explains the host cell productivity, but total profile reflects their viability and growth.•Using time-course morphological profile, antibody productivity can be predicted.•Using time-point morphological profile at day 3, productivity at day 8 can be predicted.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•Mechanistic model developed for acoustic wave separation using population balance model.•Acoustic agglomeration kernel predicts CHO cell size distribution as a function of time.•Cell trajectory has ...been analysed numerically based on the inertial and acoustic forces.•Significant effect of flowrate and acoustic power on cell dynamics in the acoustic zone.•A generalized correlation is developed using Buckingham Pi theorem to predict agglomeration efficiency.
Acoustic wave separation (AWS) is gradually emerging as an effective method for continuous clarification of Chinese hamster ovary (CHO) cells. In this paper, we present a mechanistic model for AWS based on population balance model (PBM) along with acoustic agglomeration kernel to predict cell size distribution as a function of time. Results show that more than 90% CSE was achieved for flowrates <5 mL min−1 and acoustic power >12.5 W. Experimental results confirm that the mechanistic model offers an accurate prediction of the CHO agglomeration process. Furthermore, a generalized correlation has been developed using the Buckingham Pi theorem between the dimensionless numbers composed of relevant process and geometrical variables to predict the agglomeration efficiency. To our knowledge, this is the first such study to model the agglomeration dynamics of CHO cells in acoustic standing waves.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Nanomaterials are the sixth most emerging contaminants that are entering into aquatic habitat posing a risk to the inhabiting organisms. Nanoparticles of copper ferrite have been extensively used in ...biomedical applications. However, very limited studies are available on the cytotoxicity evaluation of copper ferrite nanoparticles (CuFe2O4NPs) on different cell lines. The current work investigates on the cytotoxicity, oxidative stress and morphological variations triggered by CuFe2O4NPs in Channel catfish ovary (CCO) cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), neutral red uptake (NRU), lipid peroxidation (LPO), catalase (CAT), reduced glutathione (GSH), glutathione sulfotransferase (GST) and glutathione peroxidase (GPX) assays after 24 h of treatment. Dose dependent decline in cell survival was noticed in MTT and NRU assays. A significant increase in LPO, GST and GPX was observed in CCO cells exposed to CuFe2O4NPs after 24 h of treatment. However, the CAT and GSH levels in CCO cells exposed to CuFe2O4NPs decreased significantly after 24 h. The CCO cells exposed to 10 μg/mL concentration of CuFe2O4NPs for 24 h showed remarkable changes in their morphology. Further, the study also describes the detailed mechanism of toxicity of CuFe2O4NPs in other model cell lines to probe the risk of inhabiting organisms.
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•The cytotoxicity of CuFe2O4NPs was evaluated using CCO cells.•MTT and NRU assays revealed CuFe2O4NPs are highly toxic to CCO cells.•CuFe2O4NPs induced morphological changes in CCO cells cell after treatment.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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