Chinese hamster ovary (CHO) cells require cysteine for growth and productivity in fed-batch cultures. In intensified processes, supplementation of cysteine at high concentrations is a challenge due ...to its limited solubility and instability in solution. Methionine can be converted to cysteine (CYS) but key enzymes, cystathionine beta-synthase (Cbs) and cystathionine gamma-lyase (Cth), are not active in CHO cells resulting in accumulation of an intermediate, homocysteine (HCY), in cell culture milieu. In this study, Cbs and Cth were overexpressed in CHO cells to confer cysteine prototrophy, i.e., the ability to grow in a cysteine free environment. These pools (CbCt) needed homocysteine and beta-mercaptoethanol (βME) to grow in CYS-free medium. To increase intracellular homocysteine levels, Gnmt was overexpressed in CbCt pools. The resultant cell pools (GnCbCt), post adaptation in CYS-free medium with decreasing residual HCY and βME levels, were able to proliferate in the HCY-free, βME-free and CYS-free environment. Interestingly, CbCt pools were also able to be adapted to grow in HCY-free and CYS-free conditions, albeit at significantly higher doubling times than GnCbCt cells, but couldn’t completely adapt to βME-free conditions. Further, single cell clones derived from the GnCbCt cell pool had a wide range in expression levels of Cbs, Cth and Gnmt and, when cultivated in CYS-free fed-batch conditions, performed similarly to the wild type (WT) cell line cultivated in CYS supplemented fed-batch culture. Intracellular metabolomic analysis showed that HCY and glutathione (GSH) levels were lower in the CbCt pool in CYS-free conditions but were restored closer to WT levels in the GnCbCt cells cultivated in CYS-free conditions. Transcriptomic analysis showed that GnCbCt cells upregulated several genes encoding transporters as well as methionine catabolism and transsulfuration pathway enzymes that support these cells to biosynthesize cysteine effectively. Further, ‘omics analysis suggested CbCt pool was under ferroptotic stress in CYS-free conditions, which, when inhibited, enhanced the growth and viability of these cells in CYS-free conditions.
•Cysteine is difficult to supplement & control in CHO cell processes•Cth, Cbs, & Gnmt (GnCbCt) or Cth & Cbs (CbCt) overexpression confers CYS prototrophy•In -CYS, GnCbCt grows better than CbCt & produce titers comparable to WT in +CYS•GnCbCt cells have higher intracellular CYS than CbCt but are comparable to WT levels•GnCbCt express other genes that further enable growth in CYS-free conditions•CbCt cells appear to be under ferroptotic stress but not GnCbCt cells
Cytotoxic T-lymphocyte-associated protein 4-Ig (CTLA4-Ig) produced using Chinese hamster ovary (CHO) cell lines is a fusion protein of CTLA4 and the Fc region of antibody. In the present study, we ...identified and overexpressed genes capable of increasing sialic acid levels in CTLA4-Ig to develop cell lines using glycoengineering technology. CTLA4-Ig was produced using CHO cells overexpressing N-acetylglucosaminyltransferase (GnT) and α2,6-sialyltransferase (α2,6-ST). The conditions were wild type (WT), overexpression (GnT-IV, GnT-V, and α2,6-ST), and co-overexpression (GnT-IV and α2,6-ST, and GnT-V and α2,6-ST). GnT-IV and GnT-V were transfected into CHO cells to determine tri-antennary structure formation in CTLA4-Ig. CHOGnT-IV (cells overexpressing GnT-IV) showed the highest tri-antennary structures of glycans. Compared to CHOWT, neutral and mono-sialylated glycans decreased (−10.9% and −18.6%, respectively), while bi- and tri-sialylated N-glycans increased (4.1% and 85.7%, respectively) in CHOGnT-IV∙ST (cells co-overexpressing GnT-IV and α2,6-ST). The sum of the relative quantities of neutral N-glycans decreased from 32.0% to 28.5%, while that of sialylated N-glycans increased from 68.0% to 71.5% in CHOGnT-IV∙ST. These results are the first to demonstrate the co-overexpression of especially GnT-IV and α2,6-ST, which is an effective strategy to increase sialic acid levels and the tri-antennary structure of CTLA4-Ig produced using CHO cell lines.
•Protein-specific glycoengineered CHO cells were constructed.•Overexpression of GnT-IV played a major role in antennary structure formation of CTLA4-Ig.•Co-overexpression of GnT-IV and α2,6-ST made highly sialylated CTLA4-Ig.
Due to the favorable attributes of Chinese hamster ovary (CHO) cells for therapeutic proteins and antibodies biomanufacturing, companies generate proprietary cells with desirable phenotypes. One key ...attribute is the ability to stably express multi‐gram per liter titers in chemically defined media. Cell, media, and feed diversity has limited community efforts to translate knowledge. Moreover, academic, and nonprofit researchers generally cannot study “industrially relevant” CHO cells due to limited public availability, and the time and knowledge required to generate such cells. To address these issues, a university‐industrial consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) has acquired two CHO “reference cell lines” from different lineages that express monoclonal antibodies. These reference cell lines have relevant production titers, key performance outcomes confirmed by multiple laboratories, and a detailed technology transfer protocol. In commercial media, titers over 2 g/L are reached. Fed‐batch cultivation data from shake flask and scaled‐down bioreactors is presented. Using productivity as the primary attribute, two academic sites aligned with tight reproducibility at each site. Further, a chemically defined media formulation was developed and evaluated in parallel to the commercial media. The goal of this work is to provide a universal, industrially relevant CHO culture platform to accelerate biomanufacturing innovation.
In this work, Cordova and coauthors present a CHO platform process using non‐proprietary cells, protocols, and chemically‐defined growth media and feeds with industrially‐relevant performance. The performance of the system was tested at two independent sites for growth in shake flasks and scale‐down bioreactors. Perhaps was comparable across the sites and conditions tested.
The biopharmaceutical industry extensively employs Chinese hamster ovary (CHO) cell culture for monoclonal antibody production. Amino acids represent an essential source of nutrients in all CHO cell ...culture media, and their concentration is known to significantly impact cell viability, titre, and monoclonal antibody critical quality attributes. In this study, a robust Fourier transform near-infrared spectroscopy (FT-NIR) based quantification method has been developed for of all 20 amino acids (0–24 mM), as well as concentrations of glucose (0–6.7 mg mL−1), lactate (0–2.7 mg mL−1), and trastuzumab (0–2.5 mg mL−1) in the CHO cell culture. Near infra-red absorbance spectrum in the range of 4000–11,000 cm−1 were acquired, and spectra pre-processing through smoothening and derivatives were employed to enhance key characteristic signals. High-performance liquid chromatography with pre-column derivatization was used as the orthogonal analytical tool for quantification. Principal component analysis and partial least squares regression were employed for region selection and calibration model development, respectively. The results demonstrate that a good calibration statistic with the acceptable coefficient of determinations for both calibration (Rc2 = 0.94–0.99) and prediction (Rp2 = 0.83–0.98) could be achieved, along with high RPD values (>3) for all components except alanine (2.4). The external validation study also exhibited a satisfactory outcome (REV2 = 0.89–0.99, RMSE = 0.04–1.04), validating the model's ability to predict the concentrations of the respective species. The calibration models were successfully applied for at-line monitoring of two perfusion runs on a 10 L scale. To our knowledge, this is the first application where NIR spectroscopy-based measurement of all 20 amino acids in mammalian cell culture samples has been demonstrated. The proposed tool can play a critical role as biopharma manufacturers implement continuous processing as well as for facilitating process analytical technology-based control of mammalian cell culture processes.
Quantification, calibration, and real-time monitoring of media components in mammalian cell culture using NIR spectroscopy. Display omitted
•Near-infrared spectroscopy-based media quantification method developed for mammalian cell culture.•Proposed method can analyze all 20 amino acids, glucose, lactate and trastuzumab within realistic operational ranges.•Spectra pre-processing through smoothening and derivatives were employed to enhance key characteristic signal.•Excellent prediction statistics was achieved for both calibration (Rc2 = 0.94–0.99) and prediction (Rp2 = 0.83–0.98) dataset.
Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant protein, fed-batch culture is ...typically used for recombinant protein production in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch optimization should have a connection with comprehensive factors such as culture environmental parameters, feed composition, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and the intensified fed-batch are reviewed.
Key points
• Fed-batch culture in CHO cells is reviewed.
• Fed-batch has become a common technology for recombinant protein production.
• Fed batch culture promotes recombinant protein production in CHO cells.
Coagulation with or without pre-oxidation are important drinking water treatment processes. However, the efficacy of these processes in mitigating water toxicity remains unknown. To further improve ...drinking water safety, we employed water from the Pearl River Delta region of southern China to investigate a treatment approach consisting of coagulation with or without pre-oxidation to simultaneously modulate health-relevant cytotoxicity to CHO cells, on top of the conventional foci of turbidity and dissolved organic carbon (DOC) during water treatment. Three coagulants (two aluminum-based and one iron-based salts) and three pre-oxidants (ozone, permanganate, and peroxymonosulfate) were studied. For coagulation without pre-oxidation, intermediate coagulant doses and pH reached optimum cytotoxicity to CHO cells, turbidity, and DOC control simultaneously. Introducing oxidants reduced cytotoxicity to CHO cells significantly, enhanced by increasing oxidant concentrations and pre-oxidation duration. The cytotoxicity to CHO cells mitigation capabilities of three pre-oxidants were: ozone > peroxymonosulfate > potassium permanganate. Modulation of water cytotoxicity to CHO cells was mostly attributable to controlling DOC (specifically humic-acid like substances, tyrosine, tryptophan). However, the addition of pre-oxidants led to significant shifts in water cytotoxicity to CHO cells forcing drivers, rendering humic-acid like substances the sole decisive cytotoxicity-inducing fluorophores. For the first time, ‘sweet spots’ were identified to simultaneously monitor cytotoxicity to CHO cells alongside turbidity and DOC. These methods better modulate water cytotoxicity to CHO cells without sacrificing conventional water treatment goals while shedding light onto the mechanisms behind.
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•Addition of pre-oxidants led to shifts in toxicity drivers.•‘Sweet spot’ was identified for simultaneous control of toxicity, turbidity, and DOC.•Treatments incorporating PAC exhibited lowest cytotoxicity.•Pre-oxidation with O3 coupled to coagulation with PAC produced lowest cytotoxicity.•Toxicity control was mainly attributed to modulation of humic acid-like substances.
Supply and uptake of amino acids is of great importance to mammalian cell culture processes. Mammalian cells such as Chinese hamster ovary (CHO) cells express several amino acid (AA) transporters ...including uniporters and exchangers. Each transporter transports multiple AAs, making prediction of the effect of changed medium composition or transporter levels on individual AA transport rate challenging. A general kinetic model for such combinatorial amino acid transport, and a simplified analytical expression for the uptake rate as a function of amino acid concentrations and transporter levels is presented. From this general model, a CHO cell-specific AA transport model, to our knowledge the first such network model for any cell type, is constructed. The model is validated by its prediction of reported uptake flux and dependencies from experiments that were not used in model construction or parameter estimation. The model defines theoretical conditions for synergistic/repressive effect on the uptake rates of other AAs upon external addition of one AA. The ability of the CHO-specific model to predict amino acid interdependencies experimentally observed in other mammalian cell types suggests its robustness. This model will help formulate testable hypotheses of the effect of process changes on AA initial uptake, and serve as the AA transport component of kinetic models for cellular metabolism.
•First model for multi-amino acid multi-type-transporter system in CHO cells.•Analytical expression for estimating amino acid uptake rates from concentration data.•Predicts experimentally reported uptake rate from data not used in model formulation.•Predicts change in one amino acid uptake rate upon addition of another amino acid.•Modular formulation will enable use in comprehensive metabolism models.
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