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Mammalian cells are commonly used as hosts in cell culture for biologics production in the pharmaceutical industry. Structured mechanistic models of metabolism have been used to capture complex cellular mechanisms that contribute to varying metabolic shifts in different cell lines. However, little research has focused on the impact of temporal changes in enzyme abundance and activity on the modeling of cell metabolism. In this work, we present a framework for constructing mechanistic models of metabolism that integrate growth‐signaling control of enzyme activity and transcript dynamics. The proposed approach is applied to build models for three Chinese hamster ovary (CHO) cell lines using fed‐batch culture data and time‐series transcript profiles. Leveraging information from the transcriptome data, we develop a parameter estimation approach based on multi‐cell‐line (MCL) learning, which combines data sets from different cell lines and trains the individual cell‐line models jointly to improve model accuracy. The computational results demonstrate the important role of growth signaling and transcript variability in metabolic models as well as the virtue of the MCL approach for constructing cell‐line models with a limited amount of data. The resulting models exhibit a high level of accuracy in predicting distinct metabolic behaviors in the different cell lines; these models can potentially be used to accelerate the process and cell‐line development for the biomanufacturing of new protein therapeutics. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) used in biomedical research and therapeutic applications are primarily produced by Chinese hamster ovary (CHO) cells via fed-batch culture. The growing need for elevated quantities of biologics mandates the continual optimization of the mAb production process. The development of an effective process control method is indispensable for the production of specified mAbs by CHO cells. In this study, the effects of the pH control strategy on CHO cell fed-batch culture to produce an antibody (EA5) were first investigated in a 3 L bioreactor. The results indicate that controlling the culture pH at 7.2 during the fed-batch stage could produce a higher EA5 titer of 6.1 g/L with a lower Man5 ratio of 2.2% by day 14. Based on this, an optimized CHO cell fed-batch culture was conducted in a 15 L bioreactor to verify its effectiveness and stability. In this case, on day 14, an EA5 titer of 6.5 g/L was achieved with productivity of 0.46 g/L/day, which was 1.07-fold higher compared to that of the culture in the 3 L bioreactor. Furthermore, regarding the product quality, a monomer abundance of 96.0%, a main peak of 55.0%, and a Man5 proportion of 2.4% were maintained in the 15 L bioreactor. In addition, different cell clarification processes were evaluated using the CHO cell culture broth from the 3 L and 15 L bioreactors to further improve productivity and economic performance. Overall, this study provides some directions for process intensification and improving the quality of mAbs produced by CHO cells in the biopharmaceutical industry. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hollow fiber filter fouling is a common issue plaguing perfusion production process for biologics therapeutics, but the nature of filter foulant has been elusive. Here we studied cell culture materials especially Chinese hamster ovary (CHO) cell‐derived extracellular vesicles in perfusion process to determine their role in filter fouling. We found that the decrease of CHO‐derived small extracellular vesicles (sEVs) with 50–200 nm in diameter in perfusion permeates always preceded the increase in transmembrane pressure (TMP) and subsequent decrease in product sieving, suggesting that sEVs might have been retained inside filters and contributed to filter fouling. Using scanning electron microscopy and helium ion microscopy, we found sEV‐like structures in pores and on foulant patches of hollow fiber tangential flow filtration filter (HF‐TFF) membranes. We also observed that the Day 28 TMP of perfusion culture correlated positively with the percentage of foulant patch areas. In addition, energy dispersive X‐ray spectroscopy‐based elemental mapping microscopy and spectroscopy analysis suggests that foulant patches had enriched cellular materials but not antifoam. Fluorescent staining results further indicate that these cellular materials could be DNA, proteins, and even adherent CHO cells. Lastly, in a small‐scale HF‐TFF model, addition of CHO‐specific sEVs in CHO culture simulated filter fouling behaviors in a concentration‐dependent manner. Based on these results, we proposed a mechanism of HF‐TFF fouling, in which filter pore constriction by CHO sEVs is followed by cake formation of cellular materials on filter membrane. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) used in biomedical research and therapeutic applications are primarily produced by Chinese hamster ovary (CHO) cells via fed-batch culture. The growing need for elevated quantities of biologics mandates the continual optimization of the mAb production process. The development of an effective process control method is indispensable for the production of specified mAbs by CHO cells. In this study, the effects of the pH control strategy on CHO cell fed-batch culture to produce an antibody (EA5) were first investigated in a 3 L bioreactor. The results indicate that controlling the culture pH at 7.2 during the fed-batch stage could produce a higher EA5 titer of 6.1 g/L with a lower Man5 ratio of 2.2% by day 14. Based on this, an optimized CHO cell fed-batch culture was conducted in a 15 L bioreactor to verify its effectiveness and stability. In this case, on day 14, an EA5 titer of 6.5 g/L was achieved with productivity of 0.46 g/L/day, which was 1.07-fold higher compared to that of the culture in the 3 L bioreactor. Furthermore, regarding the product quality, a monomer abundance of 96.0%, a main peak of 55.0%, and a Man5 proportion of 2.4% were maintained in the 15 L bioreactor. In addition, different cell clarification processes were evaluated using the CHO cell culture broth from the 3 L and 15 L bioreactors to further improve productivity and economic performance. Overall, this study provides some directions for process intensification and improving the quality of mAbs produced by CHO cells in the biopharmaceutical industry.

Invasive Aspergillosis is a high-risk illness with a high death rate in immunocompromised people due to a lack of early detection and timely treatment. Based on immunology study, we achieved an efficient production of anti-galactomannan antibody by Chinese hamster ovary (CHO) cells and applied it to time-resolved fluoroimmunoassay for Aspergillus galactomannan detection. We first introduced dual promoter expression vector into CHO host cells, and then applied a two-step screening strategy to screen the stable cell line by methionine sulfoximine pressurization. After amplification and fermentation, antibody yield reached 4500 mg/L. Then we conjugated the antibodies with fluorescent microspheres to establish a double antibody sandwich time-resolved fluoroimmunoassay, which was compared with the commercial Platelia™ Aspergillus Ag by clinical serum samples. The preformed assay could obtain the results in less than 25 min, with a limit of detection for galactomannan of approximately 1 ng/mL. Clinical results of the two methods showed that the overall percent agreement was 97.7% (95% CI: 96.6%–98.4%) and Cohen's kappa coefficient was 0.94. Overall, the assay is highly consistent with commercial detection, providing a more sensitive and effective method for the rapid diagnosis of invasive aspergillosis. [Display omitted] • An efficient and stable cell line expression system was constructed to produce of anti-galactomannan monoclonal antibodies. • The antibody yield reached 4500 mg/L by scaling up fermentation, a 1.3-fold increase. • A time-resolved fluorescence immunoassay was established by conjugating the antibodies to detect aspergillosis. • The assay is highly consistent with commercial Platelia™ Aspergillus Ag according to clinical serum results. [ABSTRACT FROM AUTHOR]

Rapidly expanding pipelines in biopharmaceutical industry and increasingly stringent requirements from regulatory agencies call for a more efficient and rational study design at early-stage cell culture process development. Recent developments in high-throughput bioreactor system and design of experiments (DoE) enable cell culture process development sector to meet such demand by integrating both technological advances. In this study, we compared varied forms of fractional factorial designs (FFDs) and definitive screening design (DSD) covering key process parameters such as temperature shift target, temperature shift timing, feed strategy, pH, dissolved oxygen, harvest time and additives in Ambr® 15 bioreactors to better understand the initial cell culture process and thus optimize it. Results showed that DSD was more effective and efficient in detecting both main and quadratic effects comparing to FFDs. Comparison of varied available model development strategies revealed that two-stage forward regression combined with corrected Akaike's information criterion (AICc) and least absolute shrinkage and selection operator (LASSO) regression combined with self-validated ensembled models (SVEM) offered superior predictive capability. Moreover, key DoE regularities was evaluated for their validity. In conclusion, DSD is a suitable experimental design for the early-stage cell culture process development if quadratic effects might exist. Statistical models derived based on DSD-based dataset could be applied for predictive optimization of antibody producing bioprocess while regularities such as effect heredity and effect hierarchy might be relaxed considering higher-order interaction effects are more likely to existed for a cell-based biological system. • Utilizing high-throughput bioreactors and DoE to streamline early-stage cell culture optimization. • DSD was more effective and efficient in detecting both main and quadratic effects comparing to FFDs. • AICc-based two-stage forward stepwise regression models and SVEM-Lasso offered superior predictive capability. • Relaxing effect heredity and hierarchy while still adhering to the principles of effect sparsity and model parsimony. [ABSTRACT FROM AUTHOR]