Chinese Hamster Ovary Cell Culture Research Articles

Effect of 4-phenylbutyrate addition timing on titer of Fc-fusion protein in Chinese hamster ovary cell cultures

Effect of 4-phenylbutyrate addition timing on titer of Fc-fusion protein in Chinese hamster ovary cell cultures

In-line coupling of capillary-channeled polymer fiber columns with optical absorbance and multi-angle light scattering detection for the isolation and characterization of exosomes

Extracellular vesicles (EVs) have garnered much interest due to their fundamental role in intracellular communication and their potential utility in clinical diagnostics and as biotherapeutic vectors. Of particular relevance is the subset of EVs referred to as exosomes, ranging in size from 30 to 150 nm, which contain incredible amounts of information about their cell of origin, which can be used to track the progress of disease. As a complementary action, exosomes can be engineered with therapeutic cargo to selectively target diseases. At present, the lack of highly efficient methods of isolation/purification of exosomes from diverse biofluids, plants, and cell cultures is a major bottleneck in the fundamental biochemistry, clinical analysis, and therapeutic applications. Equally impactful, the lack of effective in-line means of detection/characterization of isolate populations, including concentration and sizing, is limiting in the applications. The method presented here couples hydrophobic interaction chromatography (HIC) performed on polyester capillary-channeled polymer (C-CP) fiber columns followed by in-line optical absorbance and multi-angle light scattering (MALS) detection for the isolation and characterization of EVs, in this case present in the supernatant of Chinese hamster ovary (CHO) cell cultures. Excellent correlation was observed between the determined particle concentrations for the two detection methods. C-CP fiber columns provide a low-cost platform (< $5 per column) for the isolation of exosomes in a 15-min workflow, with complementary absorbance and MALS detection providing very high-quality particle concentration and sizing information.

Measurement and control of foam generation in a mammalian cell culture.

Foam is generated in mammalian cell cultures by excessive agitation or gas sparging. This occurs particularly in cultures that generate recombinant proteins at high cell concentrations. Three antifoam agents were tested for their compatibility with antibody-producing Chinese hamster ovary (CHO) cells. One agent (antifoam 204) was completely inhibitory to growth at a concentration of 10 ppm, one agent (antifoam C) showed partial inhibition and a third (antifoam SE-15) showed no inhibition at this concentration. A novel foam image analyzer (LabCam) was used to evaluate two antifoams (C and SE-15) for their ability to dissipate foam generated in cell culture media by enhanced agitation. The presence of antifoam in the media reduced significantly the foam layer that was generated and this was shown to be rapidly dissipated in the presence of 10 ppm SE-15. The antifoams were also tested for foam dissipation in cultures of CHO cells at >106 cells/mL. Supplementation of the cultures with SE-15 resulted in dissipation of foam generated by excessive gas sparging within 2 min. Under equivalent conditions 75% of foam dissipated in the presence of antifoam C, within 2 min but there was a residual foam layer up to 25 min. This study showed the value of an optical monitoring system (LabCam) for measuring foam generation and dissipation in a bioreactor to assess the efficiency of antifoam agents to reduce foam in a bioreactor. This has the potential for use as a control system that could be designed for continuous monitoring and foam control in a mammalian cell bioprocess.

Elucidating uptake and metabolic fate of dipeptides in CHO cell cultures using 13C labeling experiments and kinetic modeling

The rapidly growing market of biologics including monoclonal antibodies has stimulated the need to improve biomanufacturing processes including mammalian host systems such as Chinese Hamster Ovary (CHO) cells. Cell culture media formulations continue to be enhanced to enable intensified cell culture processes and optimize cell culture performance. Amino acids, major components of cell culture media, are consumed in large amounts by CHO cells. Due to their low solubility and poor stability, certain amino acids including tyrosine, leucine, and phenylalanine can pose major challenges leading to suboptimal bioprocess performance. Dipeptides have the potential to replace amino acids in culture media. However, very little is known about the cleavage, uptake, and utilization kinetics of dipeptides in CHO cell cultures. In this study, replacing amino acids, including leucine and tyrosine by their respective dipeptides including but not limited to Ala-Leu and Gly-Tyr, supported similar cell growth, antibody production, and lactate profiles. Using 13C labeling techniques and spent media studies, dipeptides were shown to undergo both intracellular and extracellular cleavage in cultures. Extracellular cleavage increased with the culture duration, indicating cleavage by host cell proteins that are likely secreted and accumulate in cell culture over time. A kinetic model was built and for the first time, integrated with 13C labeling experiments to estimate dipeptide utilization rates, in CHO cell cultures. Dipeptides with alanine at the N-terminus had a higher utilization rate than dipeptides with alanine at the C-terminus and dipeptides with glycine instead of alanine at N-terminus. Simultaneous supplementation of more than one dipeptide in culture led to reduction in individual dipeptide utilization rates indicating that dipeptides compete for the same cleavage enzymes, transporters, or both. Dipeptide utilization rates in culture and cleavage rates in cell-free experiments appeared to follow Michaelis-Menten kinetics, reaching a maximum at higher dipeptide concentrations. Dipeptide utilization behavior was found to be similar in cell-free and cell culture environments, paving the way for future testing approaches for dipeptides in cell-free environments prior to use in large-scale bioreactors. Thus, this study provides a deeper understanding of the fate of dipeptides in CHO cell cultures through an integration of cell culture, 13C labeling, and kinetic modeling approaches providing insights in how to best use dipeptides in media formulations for robust and optimal mammalian cell culture performance.

Catechins prevent monoclonal antibody fragmentation during production via fed-batch culture of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are widely used for the industrial production of therapeutic monoclonal antibodies (mAbs). To meet the increasing market demands, high productivity, and quality are required in cell culture. One of the critical attributes of mAbs, from a safety perspective, is mAb fragmentation. However, methods for preventing mAbs fragmentation in CHO cell culture are limited. In this study, we observed that the antibody fragment content increased with increasing titers in fed-batch cultures for all three cell lines expressing recombinant antibodies. Adding copper sulfate to the culture medium further increased the fragment content, suggesting the involvement of reactive oxygen species (ROS) in the fragmentation process. Though antioxidants may be helpful to scavenge ROS, several antioxidants are reported to decrease the productivity of CHO cells. Among the antioxidants examined, we observed that the addition of catechin or (-)-epigallocatechin gallate to the culture medium prevented fragmentation content by about 20% and increased viable cell density and titer by 30% and 10%, respectively. Thus, the addition of catechins or compounds of equivalent function would be beneficial for manufacturing therapeutic mAbs with a balance between high titers and good quality.

Effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoprotein in recombinant CHO cells

The occurrence of autophagy in recombinant Chinese hamster ovary (rCHO) cell culture has attracted attention due to its effects on therapeutic protein production. Given the significance of glycosylation in therapeutic proteins, this study examined the effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoproteins in rCHO cells. Three chemical autophagy inhibitors known to inhibit different stages were separately treated with two rCHO cell lines that produce the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44. All autophagy inhibitors significantly decreased the sialylation of Fc-fusion glycoprotein in both cell lines. The decrease in sialylation of Fc-fusion glycoprotein is unlikely to be attributed to the release of intracellular enzymes, given the high cell viability and low activity of extracellular sialidases. Interestingly, the five intracellular nucleotide sugars remained abundant in cells treated with autophagy inhibitors. In the mRNA expression profiles of 27 N-glycosylation-related genes using the NanoString nCounter system, no significant differences in gene expression were noted. With the positive effect of supplementing nucleotide sugar precursors on sialylation, attempts were made to enhance the levels of intracellular nucleotide sugars by supplying these precursors. The addition of nucleotide sugar precursors to cultures treated with inhibitors successfully enhanced the sialylation of Fc-fusion glycoproteins compared to the control culture. This was particularly evident under mild stress conditions and not under relatively severe stress conditions, which were characterized by a high decrease in sialylation. These results suggest that inhibiting autophagy in rCHO cell culture decreases sialylation of Fc-fusion glycoprotein by constraining the availability of intracellular nucleotide sugars.Key points• The autophagy inhibition in rCHO cell culture leads to a significant reduction in the sialylation of Fc-fusion glycoprotein.• The pool of five intracellular nucleotide sugars remained highly abundant in cells treated with autophagy inhibitors.• Supplementation of nucleotide sugar precursors effectively restores decreased sialylation, particularly under mild stress conditions but not in relatively severe stress conditions.Graphical

In-line buffer exchange in the coupling of Protein A chromatography with weak cation exchange chromatography for the determination of charge variants of immunoglobulin G derived from chinese hamster ovary cell cultures

Immunoglobulin G (IgG) is the most common monoclonal antibody (mAb) grown for therapeutic applications. While IgG is often selectively isolated from cell lines using protein A (ProA) chromatography, this is only a stepping stone for complete characterization. Further classification can be obtained from weak cation exchange chromatography (WCX) to determine IgG charge variant distributions. The charge variants of monoclonal antibodies can influence the stability and efficacy in vivo, and deviations in charge heterogeneity are often cell-specific and sensitive to upstream process variability. Current methods to characterize IgG charge variants are often performed off-line, meaning that the IgG eluate from the ProA separation is collected, diluted to adjust the pH, and then transferred to the WCX separation, adding time, complexity, and potential contamination to the sample analysis process. More recently, reports have appeared to streamline this separation using in-line two-dimensional liquid chromatography (2D-LC). Presented here is a novel, 2D-LC coupling of ProA in the first dimension (1D) and WCX in the second dimension (2D) chromatography. As anticipated, the initial direct column coupling proved to be challenging due to the pH incompatibility between the mobile phases for the two stages. To solve the solvent compatibility issue, a size exclusion column was placed in the switching valve loop of the 2D-LC instrument to act as a means for the on-line solvent exchange. The efficacy of the methodology presented was confirmed through a charge variant determination using the NIST monoclonal antibody standard (NIST mAb), yielding correct acidic, main, and basic variant compositions. The methodology was employed to determine the charge variant profile of IgG from an in-house cultured Chinese hamster ovary (CHO) cell supernatant. It is believed that this methodology can be easily implemented to provide higher-throughput assessment of IgG charge variants for process monitoring and cell line development.

Amino acid degradation pathway inhibitory by-products trigger apoptosis in CHO cells.

Chinese hamster ovary (CHO) cells are widely used to produce complex biopharmaceuticals. Improving their productivity is necessary to fulfill the growing demand for such products. One way to enhance productivity is by cultivating cells at high densities, but inhibitory by-products, such as metabolite derivatives from amino acid degradation, can hinder achieving high cell densities. This research examines the impact of these inhibitory by-products on high-density cultures. We cultured X1 and X2 CHO cell lines in a small-scale semi-perfusion system and introduced a mix of inhibitory by-products on day 10. The X1 and X2 cell lines were chosen for their varied responses to the by-products; X2 was susceptible, while X1 survived. Proteomics revealed that the X2 cell line presented changes in the proteins linked to apoptosis regulation, cell building block synthesis, cell growth, DNA repair, and energy metabolism. We later used the AB cell line, an apoptosis-resistant cell line, to validate the results. AB behaved similar to X1 under stress. We confirmed the activation of apoptosis in X2 using a caspase assay. This research provides insights into the mechanisms of cell death triggered by inhibitory by-products and can guide the optimization of CHO cell culture for biopharmaceutical manufacturing.

Exploring metabolic effects of dipeptide feed media on CHO cell cultures by in silico model-guided flux analysis

There is a growing interest in perfusion or continuous processes to achieve higher productivity of biopharmaceuticals in mammalian cell culture, specifically Chinese hamster ovary (CHO) cells, towards advanced biomanufacturing. These intensified bioprocesses highly require concentrated feed media in order to counteract their dilution effects. However, designing such condensed media formulation poses several challenges, particularly regarding the stability and solubility of specific amino acids. To address the difficulty and complexity in relevant media development, the biopharmaceutical industry has recently suggested forming dipeptides by combining one from problematic amino acids with selected pairs to compensate for limitations. In this study, we combined one of the lead amino acids, L-tyrosine, which is known for its poor solubility in water due to its aromatic ring and hydroxyl group, with glycine as the partner, thus forming glycyl-L-tyrosine (GY) dipeptide. Subsequently, we investigated the utilization of GY dipeptide during fed-batch cultures of IgG-producing CHO cells, by changing its concentrations (0.125 × , 0.25 × , 0.5 × , 1.0 × , and 2.0 ×). Multivariate statistical analysis of culture profiles was then conducted to identify and correlate the most significant nutrients with the production, followed by in silico model-guided analysis to systematically evaluate their effects on the culture performance, and elucidate metabolic states and cellular behaviors. As such, it allowed us to explain how the cells can more efficiently utilize GY dipeptide with respect to the balance of cofactor regeneration and energy distribution for the required biomass and protein synthesis. For example, our analysis results uncovered specific amino acids (Asn and Gln) and the 0.5 × GY dipeptide in the feed medium synergistically alleviated the metabolic bottleneck, resulting in enhanced IgG titer and productivity. In the validation experiments, we tested and observed that lower levels of Asn and Gln led to decreased secretion of toxic metabolites, enhanced longevity, and elevated specific cell growth and titer.Key points• Explored the optimal Tyr dipeptide for the enhanced CHO cell culture performance• Systematically analyzed effects of dipeptide media by model-guided approach• Uncovered synergistic metabolic utilization of amino acids with dipeptideGraphical abstract

Development of an in-line Raman analytical method for commercial-scale CHO cell culture process monitoring: Influence of measurement channels and batch number on model performance.

The mammalian cell culture process is a key step in commercial therapeutic protein production and needs to be monitored and controlled due to its complexity. Raman spectroscopy has been reported for cell culture process monitoring by analysis of many important parameters. However, studies on in-line Raman monitoring of the cell culture process were mainly conducted on small or pilot scale. Developing in-line Raman analytical methods for commercial-scale cell culture process monitoring is more challenging. In this study, an in-line Raman analytical method was developed for monitoring glucose, lactate, and viable cell density (VCD) in the Chinese hamster ovary (CHO) cell culture process during commercial production of biosimilar adalimumab (1500 L). The influence of different Raman measurement channels was considered to determine whether to merge data from different channels for model development. Raman calibration models were developed and optimized, with minimum root mean square error of prediction of 0.22gL-1 for glucose in the range of 1.66-3.53gL-1 , 0.08gL-1 for lactate in the range of 0.15-1.19gL-1 , 0.31 E6 cellsmL-1 for VCD in the range of 0.96-5.68 E6 cellsmL-1 on test sets. The developed analytical method can be used for cell culture process monitoring during manufacturing and meets the analytical purpose of this study. Further, the influence of the number of batches used for model calibration on model performance was also studied to determine how many batches are needed basically for method development. The proposed Raman analytical method development strategy and considerations will be useful for monitoring of similar bioprocesses.

Comprehensive modeling of cell culture profile using Raman spectroscopy and machine learning

Chinese hamster ovary (CHO) cells are widely utilized in the production of antibody drugs. To ensure the production of large quantities of antibodies that meet the required specifications, it is crucial to monitor and control the levels of metabolites comprehensively during CHO cell culture. In recent years, continuous analysis methods employing on-line/in-line techniques using Raman spectroscopy have attracted attention. While these analytical methods can nondestructively monitor culture data, constructing a highly accurate measurement model for numerous components is time-consuming, making it challenging to implement in the rapid research and development of pharmaceutical manufacturing processes. In this study, we developed a comprehensive, simple, and automated method for constructing a Raman model of various components measured by LC–MS and other techniques using machine learning with Python. Preprocessing and spectral-range optimization of data for model construction (partial least square (PLS) regression) were automated and accelerated using Bayes optimization. Subsequently, models were constructed for each component using various model construction techniques, including linear regression, ridge regression, XGBoost, and neural network. This enabled the model accuracy to be improved compared with PLS regression. This automated approach allows continuous monitoring of various parameters for over 100 components, facilitating process optimization and process monitoring of CHO cells.

Enhancing the production of recombinant human TGF-β1 through an understanding of TGF-β1 synthesis, signaling, and endocytosis in CHO cells.

To enhance the production of recombinant human transforming growth factor-beta1 (rhTGF-β1) in Chinese hamster ovary (CHO) cells, rhTGF-β1 was first characterized for endocytosis, signaling pathway, and overall maturation process. The mature rhTGF-β1 used for clinical application was internalized into CHO cells and inhibited the growth of CHO cells in a dose-dependent manner. However, mature rhTGF-β1 was mostly produced in the form of latent rhTGF-β1 in cultures of recombinant CHO (rCHO) cells producing rhTGF-β1 (CHO-rhTGF-β1). The concentration of active mature rhTGF-β1 in the culture supernatant of CHO-rhTGF-β1 cells was not high enough to compromise yield. In addition, a significant amount of unprocessed precursors was produced by CHO-rhTGF-β1 cells. Overexpression of PACEsol, a soluble form of furin, in CHO-rhTGF-β1 cells was effective for the proteolytic cleavage of unprocessed precursors. The highest mature rhTGF-β1 concentration (6.4μgmL-1 ) was obtained with the PACEsol-expressing clone, which was approximately 45% higher than that of the parental clone (P<0.01). Thus, a comprehensive understanding of the intrinsic properties of rhTGF-β1 with respect to the overall maturation process, signaling pathway, and endocytosis is essential for effectively enhancing the production of mature rhTGF-β1 in CHO cells.

Quantitative Recoveries of Exosomes and Monoclonal Antibodies from Chinese Hamster Ovary Cell Cultures by Use of a Single, Integrated Two-Dimensional Liquid Chromatography Method.

Cultured cell lines are very commonly used for the mass production of therapeutic proteins, such as monoclonal antibodies (mAbs). In particular, Chinese hamster ovary (CHO) cell lines are widely employed due to their high tolerance to variations in experimental conditions and their ability to grow in suspension or serum free media. CHO cell lines are known for their ability to produce high titers of biotherapeutic products such as immunoglobulin G (IgG). An emergent alternative means of treating diseases, such as cancer, is the use of gene therapies, wherein genetic cargo is "packaged" in nanosized vesicular structures, referred to as "vectors". One particularly attractive vector option is extracellular vesicles (EVs), of which exosomes are of greatest interest. While exosomes can be harvested from virtually any human body fluid, bovine milk, or even plants, their production in cell cultures is an attractive commercial approach. In fact, the same CHO cell types employed for mAb production also produce exosomes as a natural byproduct. Here, we describe a single integrated 2D liquid chromatography (2DLC) method for the quantitative recovery of both exosomes and antibodies from a singular sample aliquot. At the heart of the method is the use of polyester capillary-channeled polymer (C-CP) fibers as the first dimension column, wherein exosomes/EVs are captured from the supernatant sample and subsequently determined by multiangle light scattering (MALS), while the mAbs are captured, eluted, and quantified using a protein A-modified C-CP fiber column in the second dimension, all in a 10 min workflow. These efforts demonstrate the versatility of the C-CP fiber phases with the capacity to harvest both forms of therapeutics from a single bioreactor, suggesting an appreciable potential impact in the field of biotherapeutics production.

Alleviation of the necessity for supernatant prefiltering in the protein a recovery of Monoclonal antibodies from Chinese hamster ovary cell cultures

Protein A (ProA) chromatography is a mainstay in the analytical and preparative scale isolation/purification of monoclonal antibodies (mAbs). One area of interest is continuous processing or continuous chromatography, where ProA chromatography is used in the large-scale purification of mAbs. However, filtration is required prior to all ProA isolations to remove large particulates in cell culture supernatant, consisting of a mixture of cell debris, host cell contaminants, media components, etc. Currently, in-line filters are used to remove particles in the supernatant, requiring replacement over time due to fouling; regardless of the scale. Here we demonstrate the ProA isolation of unfiltered Chinese hamster ovary (CHO) cell media using capillary-channel polymer (C-CP) fiber stationary phases modified with S. aureus Protein A (rSPA). The base polymer of the analytical scale C-CP columns costs ∼$5 per 30 cm column, and when modified with ProA, the base cost is ∼$25 per 30 cm column, a cost-effective option in comparison to analytical-scale commercial columns. To directly sample unfiltered media, a 5 cm gap was created at the head of the C-CP column, where the large particulates are trapped, while molecular solutes flow through the capillary channels without sacrifice in analytical performance, mAb loading capacity, or backpressure increases. The binding capacity of the gap ProA C-CP column was ∼ 2 mg mL−1 of IgG per bed volume. The same analytical column could be operated after processing a total of ∼ 56 column bed volumes of supernatant (>25 analytical cycles) without the need for caustic clean-in-place processing.

Construction of a novel kinetic model for the production process of a CVA6 VLP vaccine in CHO cells.

The online version contains supplementary material available at 10.1007/s10616-023-00598-8.

Quantitative analysis of residual aurintricarboxylic acid in biotherapeutic process streams using liquid chromatography triple quadrupole mass spectrometry

Aurintricarboxylic acid (ATA) is an excipient that can be added to the therapeutic protein manufacturing process as a component of the Chinese hamster ovary (CHO) cell culture media. ATA inhibits cell apoptosis and promotes cell growth in both serum-free and protein-free media. The addition of ATA is beneficial to the manufacturing process at the cell growth stage, however, residual ATA not consumed by cells can have toxicological effects on patients and its removal is required during downstream processing. To ensure manufacturing control and patient safety, the determination of residual ATA during downstream processing of biotherapeutics is required. The aim of this study was to develop and validate a liquid chromatography triple quadrupole mass spectrometry method for monitoring process clearance of ATA during downstream processing. Chromatographic separation of ATA was achieved using a 50 × 3 mm, 3 µm Imtakt Cadenza HS-C18 column and a triple quadrupole mass spectrometer operated in selected reaction monitoring mode was used for sensitive and specific detection. Linearity was assessed over a range of 62.5 ng/mL to 2000 ng/mL. Accuracy and precision were within 20% of the theoretical spike levels across the three concentration levels evaluated utilising two different antibodies, an IgG1 and an IgG4. Analyte specificity and selectivity were deemed acceptable based on no extraneous peaks detected. The method was successfully applied to monitoring process clearance of ATA in a tangential flow filtration (TFF) unit operation for downstream processing of monoclonal antibodies.

Continuous Online Titer Monitoring in CHO Cell Culture Supernatant Using a Herringbone Nanofluidic Filter Array.

Online monitoring of monoclonal antibody product titers throughout biologics process development and production enables rapid bioprocess decision-making and process optimization. Conventional analytical methods, including high-performance liquid chromatography and turbidimetry, typically require interfacing with an automated sampling system capable of online sampling and fractionation, which suffers from increased cost, a higher risk of failure, and a higher mechanical complexity of the system. In this study, a novel nanofluidic system for continuous direct (no sample preparation) IgG titer measurements was investigated. Tumor necrosis factor α (TNF-α), conjugated with fluorophores, was utilized as a selective binder for adalimumab in the unprocessed cell culture supernatant. The nanofluidic device can separate the bound complex from unbound TNF-α and selectively concentrate the bound complex for high-sensitivity detection. Based on the fluorescence intensity from the concentrated bound complex, a fluorescence intensity versus titer curve can be generated, which was used to determine the titer of samples from filtered, unpurified Chinese hamster ovary cell cultures continuously. The system performed direct monitoring of IgG titers with nanomolar resolution and showed a good correlation with the biolayer interferometry assays. Furthermore, by variation of the concentration of the indicator (TNF-α), the dynamic range of the system can be tuned and further expanded.

Removal of cell clusters from CHO suspension cultures based on large-particle trapping effect in spiral inertial microfluidics

Suspension cultures of Chinese hamster ovary (CHO) cells are extensively employed in biopharmaceutical production. During cultivation, CHO cell clusters often form due to cell death and other irregularities, substantially reducing cell growth and productivity. In this study, we introduce a novel method for the selective removal of large CHO cell clusters from bioreactors, utilizing the large-particle trapping effect in spiral inertial microfluidics. A single spiral inertial microfluidic device effectively removed large clusters from CHO cell culture, demonstrating high volumetric throughput, removal efficiency, and biocompatibility. This straightforward, agent-free, and continuous-flow approach can be applied to remove large mammalian cell clusters across various scales and modes of operation (discontinuous or continuous) during cell line development, bioprocess optimization, and biomanufacturing.

Debottlenecking and reformulating feed media for improved CHO cell growth and titer by data-driven and model-guided analyses.

Designing and selecting cell culture media along with their feeding are a key strategy to maximize culture performance in biopharmaceutical processes. However, the sensitivity of mammalian cells to their culture environment necessitates specific nutritional requirements for their growth and the production of high-quality proteins such as antibodies, depending on the cell lines and operational conditions employed. In this regard, previously we developed a data-driven and in-silico model-guided systematic framework to investigate the effect of growth media on Chinese hamster ovary (CHO) cell culture performance, allowing us to design and reformulate basal media. To expand our exploration for media development research, we evaluated two chemically defined feed media, A and B, using a monoclonal antibody-producing CHO K1 cell line in ambr15 bioreactor runs. We observed a significant impact of the feed media on various aspects of cell culture, including growth, longevity, viability, productivity, and the production of toxic metabolites. Specifically, the concentrated feed A was inadequate in sustaining prolonged cell culture and achieving high titers when compared to feed B. Within our framework, we systematically investigated the major metabolic bottlenecks in the tricarboxylic acid cycle and relevant amino acid transferase reactions. This analysis identified target components that play a crucial role in alleviating bottlenecks and designing highly productive cell cultures, specifically the addition of glutamate to feed A and asparagine to feed B. Based on our findings, we reformulated the feeds by adjusting the amounts of the targeted amino acids and successfully validated the effectiveness of the strategy in promoting cell growth, life span, and/or titer. This article is protected by copyright. All rights reserved.

Dynamics of Amino Acid Metabolism, Gene Expression, and Circulomics in a Recombinant Chinese Hamster Ovary Cell Line Adapted to Moderate and High Levels of Extracellular Lactate.

The accumulation of metabolic wastes in cell cultures can diminish product quality, reduce productivity, and trigger apoptosis. The limitation or removal of unintended waste products from Chinese hamster ovary (CHO) cell cultures has been attempted through multiple process and genetic engineering avenues with varied levels of success. One study demonstrated a simple method to reduce lactate and ammonia production in CHO cells with adaptation to extracellular lactate; however, the mechanism behind adaptation was not certain. To address this profound gap, this study characterizes the phenotype of a recombinant CHO K-1 cell line that was gradually adapted to moderate and high levels of extracellular lactate and examines the genomic content and role of extrachromosomal circular DNA (eccDNA) and gene expression on the adaptation process. More than 500 genes were observed on eccDNAs. Notably, more than 1000 genes were observed to be differentially expressed at different levels of lactate adaptation, while only 137 genes were found to be differentially expressed between unadapted cells and cells adapted to grow in high levels of lactate; this suggests stochastic switching as a potential stress adaptation mechanism in CHO cells. Further, these data suggest alanine biosynthesis as a potential stress-mitigation mechanism for excess lactate in CHO cells.