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  • 1
    Online Resource
    Online Resource
    Combining Butyrated ManNAc with Glycoengineered CHO Cells Improves EPO Glycan Quality and Production
    Wiley ; 2019
    In:  Biotechnology Journal Vol. 14, No. 4 ( 2019-04)
    Details
    In: Biotechnology Journal, Wiley, Vol. 14, No. 4 ( 2019-04)
    Abstract: Sodium butyrate (NaBu) is not only well‐known for enhancing protein production, but also degrades glycan quality. In this study, butyrate supplied by the precursor molecule 1,3,4‐ O ‐Bu 3 ManNAc is applied to overcome the negative effects of NaBu on glycan quality while simultaneously increasing the productivity of the model recombinant erythropoietin (EPO). The beneficial impact of 1,3,4‐ O ‐Bu 3 ManNAc on EPO glycan quality, while evident in wild‐type CHO cells, is particularly pronounced in glycoengineered CHO cells with stable overexpression of β‐1,4‐ and β‐1,6‐N‐acetylglucosaminyltransferases (GnTIV and GnTV) and α‐2,6‐sialyltransferase (ST6) enzymes responsible for N‐glycan antennarity and sialylation. Supplementation of 1,3,4‐ O ‐Bu 3 ManNAc achieves approximately 30% sialylation enhancement on EPO protein in wild‐type CHO cells. Overexpression of GnTIV/GnTV/ST6 in CHO cells increases EPO sialylation about 40%. Combining 1,3,4‐ O ‐Bu 3 ManNAc treatment in glyocengineered CHO cells promotes EPO sialylation about 75% relative to EPO from wild‐type CHO cells. Moreover, a detailed mass spectrometric ESI‐LC‐MS/MS characterization of glycans at each of the three N‐glycosylation sites of EPO showed that the 1st N‐site is highly sialylated and either the negative impact of NaBu or the beneficial effect 1,3,4‐ O ‐Bu 3 ManNAc treatments mainly affects the 2nd and 3rd N‐glycan sites of EPO protein. In summary, these results demonstrate 1,3,4‐ O ‐Bu 3 ManNAc can compensate for the negative effect of NaBu on EPO glycan quality while simultaneously enhancing recombinant protein yields. In this way, a platform that integrates glycoengineering with metabolic supplementation can result in synergistic improvements in both production and glycosylation in CHO cells.
    Type of Medium: Online Resource
    ISSN: 1860-6768 , 1860-7314
    URL: Issue
    URL: Article
    DOI: 10.1002/biot.v14.4
    DOI: 10.1002/biot.201800186
    Language: English
    Publisher: Wiley
    Publication Date: 2019
    detail.hit.zdb_id: 2214038-4
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  • 2
    Online Resource
    Online Resource
    Utilizing genome-scale models to optimize nutrient supply for sustained algal growth and lipid productivity
    Springer Science and Business Media LLC ; 2019
    In:  npj Systems Biology and Applications Vol. 5, No. 1 ( 2019-09-24)
    Details
    In: npj Systems Biology and Applications, Springer Science and Business Media LLC, Vol. 5, No. 1 ( 2019-09-24)
    Abstract: Nutrient availability is critical for growth of algae and other microbes used for generating valuable biochemical products. Determining the optimal levels of nutrient supplies to cultures can eliminate feeding of excess nutrients, lowering production costs and reducing nutrient pollution into the environment. With the advent of omics and bioinformatics methods, it is now possible to construct genome-scale models that accurately describe the metabolism of microorganisms. In this study, a genome-scale model of the green alga Chlorella vulgaris ( i CZ946) was applied to predict feeding of multiple nutrients, including nitrate and glucose, under both autotrophic and heterotrophic conditions. The objective function was changed from optimizing growth to instead minimizing nitrate and glucose uptake rates, enabling predictions of feed rates for these nutrients. The metabolic model control (MMC) algorithm was validated for autotrophic growth, saving 18% nitrate while sustaining algal growth. Additionally, we obtained similar growth profiles by simultaneously controlling glucose and nitrate supplies under heterotrophic conditions for both high and low levels of glucose and nitrate. Finally, the nitrate supply was controlled in order to retain protein and chlorophyll synthesis, albeit at a lower rate, under nitrogen-limiting conditions. This model-driven cultivation strategy doubled the total volumetric yield of biomass, increased fatty acid methyl ester (FAME) yield by 61%, and enhanced lutein yield nearly 3 fold compared to nitrogen starvation. This study introduces a control methodology that integrates omics data and genome-scale models in order to optimize nutrient supplies based on the metabolic state of algal cells in different nutrient environments. This approach could transform bioprocessing control into a systems biology-based paradigm suitable for a wide range of species in order to limit nutrient inputs, reduce processing costs, and optimize biomanufacturing for the next generation of desirable biotechnology products.
    Type of Medium: Online Resource
    ISSN: 2056-7189
    URL: Article
    DOI: 10.1038/s41540-019-0110-7
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2019
    detail.hit.zdb_id: 2841868-2
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  • 3
    Online Resource
    Online Resource
    Optimization of nutrient utilization efficiency and productivity for algal cultures under light and dark cycles using genome-scale model process control
    Springer Science and Business Media LLC ; 2023
    In:  npj Systems Biology and Applications Vol. 9, No. 1 ( 2023-03-15)
    Details
    In: npj Systems Biology and Applications, Springer Science and Business Media LLC, Vol. 9, No. 1 ( 2023-03-15)
    Abstract: Algal cultivations are strongly influenced by light and dark cycles. In this study, genome-scale metabolic models were applied to optimize nutrient supply during alternating light and dark cycles of Chlorella vulgaris . This approach lowered the glucose requirement by 75% and nitrate requirement by 23%, respectively, while maintaining high final biomass densities that were more than 80% of glucose-fed heterotrophic culture. Furthermore, by strictly controlling glucose feeding during the alternating cycles based on model-input, yields of biomass, lutein, and fatty acids per gram of glucose were more than threefold higher with cycling compared to heterotrophic cultivation. Next, the model was incorporated into open-loop and closed-loop control systems and compared with traditional fed-batch systems. Closed-loop systems which incorporated a feed-optimizing algorithm increased biomass yield on glucose more than twofold compared to standard fed-batch cultures for cycling cultures. Finally, the performance was compared to conventional proportional-integral-derivative (PID) controllers. Both simulation and experimental results exhibited superior performance for genome-scale model process control (GMPC) compared to traditional PID systems, reducing the overall measured value and setpoint error by 80% over 8 h. Overall, this approach provides researchers with the capability to enhance nutrient utilization and productivity of cell factories systematically by combining genome-scale models and controllers into an integrated platform with superior performance to conventional fed-batch and PID methodologies.
    Type of Medium: Online Resource
    ISSN: 2056-7189
    URL: Article
    DOI: 10.1038/s41540-022-00260-7
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2023
    detail.hit.zdb_id: 2841868-2
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  • 4
    Online Resource
    Online Resource
    Reconstruction of reverse transsulfuration pathway enables cysteine biosynthesis and enhances resilience to oxidative stress in Chinese Hamster Ovary cells
    Elsevier BV ; 2023
    In:  Metabolic Engineering Vol. 76 ( 2023-03), p. 204-214
    Details
    In: Metabolic Engineering, Elsevier BV, Vol. 76 ( 2023-03), p. 204-214
    Type of Medium: Online Resource
    ISSN: 1096-7176
    URL: Article
    DOI: 10.1016/j.ymben.2023.02.010
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2023
    detail.hit.zdb_id: 1471017-1
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    Table3.DOCX
    Frontiers Media SA ; 2021
    In:  Frontiers in Chemistry Vol. 9 ( 2021-9-24)
    Details
    In: Frontiers in Chemistry, Frontiers Media SA, Vol. 9 ( 2021-9-24)
    Abstract: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes the extensively glycosylated spike (S) protein protruding from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the main recombinant S protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, a recombinant S protein truncated at the transmembrane domain and engineered to express a C-terminal trimerization motif was transiently produced in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on S protein, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on the glycans and glycopeptides from the S protein. The process also adds a charge to Asp and Glu which aids in ionization. We used MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to determine global and site-specific N-linked glycosylation patterns. We identified 21 and 19 out of the 22 predicted N-glycosites of the SARS-CoV-2 S proteins produced in CHO and HEK, respectively. It was found that the N-glycosite at 1,158 position (N1158) and at 122, 282 and 1,158 positions (N122, N282 and N1158) were absent on S from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human S proteins reveals that CHO-Spike exhibits more complex and higher sialylation (α2,3-linked) content while HEK-Spike exhibits more high-mannose and a small amount of α2,3- and α2,6-linked sialic acids. The N74 site represents the most abundant glycosite on both spike proteins. The relatively higher amount of high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801, and N1134) on HEK-Spike suggests that glycan-shielding may differ among the two constructs. HEK-Spike can also provide different host immune system interaction profiles based on known immune system active lectins. Collectively, these data underscore the importance of characterizing the site-specific glycosylation of recombinant human spike proteins from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in research, diagnostics and vaccines.
    Type of Medium: Online Resource
    ISSN: 2296-2646
    URL: Article
    URL: Article
    URL: Article
    URL: Article
    URL: Article
    URL: Article
    URL: Article
    URL: Article
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    URL: Article
    URL: Article
    DOI: 10.3389/fchem.2021.735558
    DOI: 10.3389/fchem.2021.735558.s001
    DOI: 10.3389/fchem.2021.735558.s002
    DOI: 10.3389/fchem.2021.735558.s003
    DOI: 10.3389/fchem.2021.735558.s004
    DOI: 10.3389/fchem.2021.735558.s005
    DOI: 10.3389/fchem.2021.735558.s006
    DOI: 10.3389/fchem.2021.735558.s007
    DOI: 10.3389/fchem.2021.735558.s008
    DOI: 10.3389/fchem.2021.735558.s009
    DOI: 10.3389/fchem.2021.735558.s010
    DOI: 10.3389/fchem.2021.735558.s011
    Language: Unknown
    Publisher: Frontiers Media SA
    Publication Date: 2021
    detail.hit.zdb_id: 2711776-5
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  • 6
    Online Resource
    Online Resource
    A genome‐scale nutrient minimization forecast algorithm for controlling essential amino acid levels in CHO cell cultures
    Wiley ; 2022
    In:  Biotechnology and Bioengineering Vol. 119, No. 2 ( 2022-02), p. 435-451
    Details
    In: Biotechnology and Bioengineering, Wiley, Vol. 119, No. 2 ( 2022-02), p. 435-451
    Abstract: Mammalian cell culture processes rely heavily on empirical knowledge in which process control remains a challenge due to the limited characterization/understanding of cell metabolism and inability to predict the cell behaviors. This study facilitates control of Chinese hamster ovary (CHO) processes through a forecast‐based feeding approach that predicts multiple essential amino acids levels in the culture from easily acquired viable cell density data. Multiple cell growth behavior forecast extrapolation approaches are considered with logistic curve fitting found to be the most effective. Next, the nutrient‐minimized CHO genome‐scale model is combined with the growth forecast model to generate essential amino acid forecast profiles of multiple CHO batch cultures. Comparison of the forecast with the measurements suggests that this algorithm can accurately predict the concentration of most essential amino acids from cell density measurement with error mitigated by incorporating off‐line amino acids concentration measurements. Finally, the forecast algorithm is applied to CHO fed‐batch cultures to support amino acid feeding control to control the concentration of essential amino acids below 1–2 mM for lysine, leucine, and valine as a model over a 9‐day fed batch culture while maintaining comparable growth behavior to an empirical‐based culture. In turn, glycine production was elevated, alanine reduced and lactate production slightly lower in control cultures due to metabolic shifts in branched‐chain amino acid degradation. With the advantage of requiring minimal measurement inputs while providing valuable and in‐advance information of the system based on growth measurements, this genome model‐based amino acid forecast algorithm represent a powerful and cost‐effective tool to facilitate enhanced control over CHO and other mammalian cell‐based bioprocesses.
    Type of Medium: Online Resource
    ISSN: 0006-3592 , 1097-0290
    URL: Issue
    URL: Article
    DOI: 10.1002/bit.v119.2
    DOI: 10.1002/bit.27994
    Language: English
    Publisher: Wiley
    Publication Date: 2022
    detail.hit.zdb_id: 1480809-2
    detail.hit.zdb_id: 280318-5
    SSG: 12
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  • 7
    Online Resource
    Online Resource
    An unconventional uptake rate objective function approach enhances applicability of genome-scale models for mammalian cells
    Springer Science and Business Media LLC ; 2019
    In:  npj Systems Biology and Applications Vol. 5, No. 1 ( 2019-07-23)
    Details
    In: npj Systems Biology and Applications, Springer Science and Business Media LLC, Vol. 5, No. 1 ( 2019-07-23)
    Abstract: Constraint-based modeling has been applied to analyze metabolism of numerous organisms via flux balance analysis and genome-scale metabolic models, including mammalian cells such as the Chinese hamster ovary (CHO) cells—the principal cell factory platform for therapeutic protein production. Unfortunately, the application of genome-scale model methodologies using the conventional biomass objective function is challenged by the presence of overly-restrictive constraints, including essential amino acid exchange fluxes that can lead to improper predictions of growth rates and intracellular flux distributions. In this study, these constraints are found to be reliably predicted by an “essential nutrient minimization” approach. After modifying these constraints with the predicted minimal uptake values, a series of unconventional objective functions are applied to minimize each individual non-essential nutrient uptake rate, revealing useful insights about metabolic exchange rates and flows across different cell lines and culture conditions. This unconventional uptake-rate objective functions (UOFs) approach is able to distinguish metabolic differences between three distinct CHO cell lines (CHO-K1, -DG44, and -S) not directly observed using the conventional biomass growth maximization solutions. Further, a comparison of model predictions with experimental data from literature correctly correlates with the specific CHO-DG44-derived cell line used experimentally, and the corresponding dual prices provide fruitful information concerning coupling relationships between nutrients. The UOFs approach is likely to be particularly suited for mammalian cells and other complex organisms which contain multiple distinct essential nutrient inputs, and may offer enhanced applicability for characterizing cell metabolism and physiology as well as media optimization and biomanufacturing control.
    Type of Medium: Online Resource
    ISSN: 2056-7189
    URL: Article
    DOI: 10.1038/s41540-019-0103-6
    Language: English
    Publisher: Springer Science and Business Media LLC
    Publication Date: 2019
    detail.hit.zdb_id: 2841868-2
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  • 8
    Online Resource
    Online Resource
    Metabolic engineering challenges of extending N-glycan pathways in Chinese hamster ovary cells
    Elsevier BV ; 2020
    In:  Metabolic Engineering Vol. 61 ( 2020-09), p. 301-314
    Details
    In: Metabolic Engineering, Elsevier BV, Vol. 61 ( 2020-09), p. 301-314
    Type of Medium: Online Resource
    ISSN: 1096-7176
    URL: Article
    DOI: 10.1016/j.ymben.2020.06.007
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2020
    detail.hit.zdb_id: 1471017-1
    SSG: 12
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  • 9
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    Online Resource
    Design and Production of Bispecific Antibodies
    MDPI AG ; 2019
    In:  Antibodies Vol. 8, No. 3 ( 2019-08-02), p. 43-
    Details
    In: Antibodies, MDPI AG, Vol. 8, No. 3 ( 2019-08-02), p. 43-
    Abstract: With the current biotherapeutic market dominated by antibody molecules, bispecific antibodies represent a key component of the next-generation of antibody therapy. Bispecific antibodies can target two different antigens at the same time, such as simultaneously binding tumor cell receptors and recruiting cytotoxic immune cells. Structural diversity has been fast-growing in the bispecific antibody field, creating a plethora of novel bispecific antibody scaffolds, which provide great functional variety. Two common formats of bispecific antibodies on the market are the single-chain variable fragment (scFv)-based (no Fc fragment) antibody and the full-length IgG-like asymmetric antibody. Unlike the conventional monoclonal antibodies, great production challenges with respect to the quantity, quality, and stability of bispecific antibodies have hampered their wider clinical application and acceptance. In this review, we focus on these two major bispecific types and describe recent advances in the design, production, and quality of these molecules, which will enable this important class of biologics to reach their therapeutic potential.
    Type of Medium: Online Resource
    ISSN: 2073-4468
    URL: Article
    DOI: 10.3390/antib8030043
    Language: English
    Publisher: MDPI AG
    Publication Date: 2019
    detail.hit.zdb_id: 2661514-9
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