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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., (© 2022 Wiley Periodicals LLC.)

Chinese hamster ovary (CHO) cell cultures in industry are most commonly conducted as fed-batch cultures in computer-controlled bioreactors, though most preliminary studies are conducted in fed-batch shake flasks. To improve comparability between bioreactor studies and shake flask studies, shake flask studies should be conducted as fed-batch. However, the smaller volumes and reduced control in shake flasks can impact pH and aeration, which leads to performance differences. Planning and awareness of these vessel and control differences can assist with experimental design as well as troubleshooting. This method will highlight several of the configuration and control issues that should be considered during the transitions from batch to fed-batch and shake flasks to bioreactors, as well as approaches to mitigate the differences. Furthermore, if significant differences occur between bioreactor and shake flask studies, approaches will be presented to isolate the main contributors for these differences., (Copyright © 2021 Elsevier Inc. All rights reserved.)

NS0 and Chinese hamster ovary (CHO) cell lines are used to produce recombinant proteins for human therapeutics; however, ammonium accumulation can negatively impact cell growth, recombinant protein production, and protein glycosylation. To improve product quality and decrease costs, the relationship between ammonium and protein glycosylation needs to be elucidated. While ammonium has been shown to adversely affect glycosylation-related gene expression in CHO cells, NS0 studies have not been performed. Therefore, this study sought to determine if glycosylation in NS0 cells were ammonium-sensitive at the gene expression level. Using a DNA microarray that contained mouse glycosylation-related and housekeeping genes, these genes were analyzed in response to various culture conditions - elevated ammonium, elevated salt, and elevated ammonium with proline. Surprisingly, no significant differences in gene expression levels were observed between the control and these conditions. Further, the elevated ammonium cultures were analyzed using real-time quantitative reverse transcriptase PCR (qRT-PCR) for key glycosylation genes, and the qRT-PCR results corroborated the DNA microarray results, demonstrating that NS0 cells are ammonium-insensitive at the gene expression level. Since NS0 are known to have elevated nucleotide sugar pools under ammonium stress, and none of the genes directly responsible for these metabolic pools were changed, consequently cellular control at the translational or substrate-level must be responsible for the universally observed decreased glycosylation quality under elevated ammonium., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Explicitly identifying the genome of a host organism including sequencing, mapping, and annotating its genetic code has become a priority in the field of biotechnology with aims at improving the efficiency and understanding of cell culture bioprocessing. Recombinant protein therapeutics, primarily produced in mammalian cells, constitute a $108 billion global market. The most common mammalian cell line used in biologic production processes is the Chinese hamster ovary (CHO) cell line, and although great improvements have been made in titer production over the past 25 years, the underlying molecular and physiological factors are not well understood. Confident understanding of CHO bioprocessing elements (e.g. cell line selection, protein production, and reproducibility of process performance and product specifications) would significantly improve with a well understood genome. This review describes mammalian cell culture use in bioprocessing, the importance of obtaining CHO cell line genetic sequences, and the current status of sequencing efforts. Furthermore, transcriptomic techniques and gene expression tools are presented, and case studies exploring genomic techniques and applications aimed to improve mammalian bioprocess performance are reviewed. Finally, future implications of genomic advances are surmised., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Protein splicing elements (inteins), capable of catalyzing controllable peptide bond cleavage reactions, have been used to separate recombinant proteins from affinity tags during affinity purification. Since the inteins eliminate the use of a protease in the recovery process, the intein-mediated purification system has the potential to significantly reduce recovery costs for the industrial production of recombinant proteins. Thus far, the intein system has only been examined and utilized for expression and purification of recombinant proteins at the laboratory scale for cells cultivated at low cell densities. In this study, protein splicing and in vitro cleavage of intein fusion proteins expressed in high-cell-density fed-batch fermentations of recombinant Escherichia coli were examined. Three model intein fusion constructs were used to examine the stability and splicing/cleavage activities of the fusion proteins produced under high-cell-density conditions. The data indicated that the intein fusion protein containing the wild-type intein catalyzed efficient in vivo protein splicing during high-cell-density cultivation. Also, the intein fusion proteins containing modified inteins catalyzed efficient thiol-induced in vitro cleavage reactions. The results of this study demonstrated the potential feasibility of using the intein-mediated protein purification system for industrial-scale production of recombinant proteins.

When working on biotherapeutic process development, the analysis of spent cell culture media is often a daily practice during the optimization of bioreactor conditions and media composition. The introduction of parallel microbioreactor systems has decreased the complexity and costs of process development by allowing for concurrent studies of multiple bioreactor and media variables. However, the bioreactors' small volumes (typically less than 250 mL) limit the volume of media one can extract for daily sampling. We describe a means to analyze spent media with an integrated microchip capillary electrophoresis mass spectrometer (CE-MS) analyzer with minimal sample volume requirements and rapid analysis time. The platform was evaluated with a parallel microbioreactor system (ambr® 250) culturing a Chinese hamster ovary (CHO) cell line stressed by varying levels of ammonia (NH3). The spent media analysis identified net increases in the levels of the amino acids (AA) Ala, Arg, Asp, Glu, Gly, His, Ile, Leu, Lys, Phe, Thr, Trp, Tyr, and Val in all bioreactors, with Gly levels showing increases in excess of 8-fold initial levels in all bioreactors. Other media components either steadily decreased in concentration or were completely depleted by the end of culture. For example, Asn was depleted in all of the unstressed and 10 mM NH3-stressed bioreactors, but was approximately twice as high as the initial levels in the 30 mM NH3-stressed bioreactors at the end of the culture periods. Also, the 30 mM NH3-stressed condition may have caused either complete degradation or rapid consumption of choline, since it was no longer present starting at the t = 36 h sampling. Overall, the monitored media components were observed to have independent trajectories based on feeding and consumption by the cells, and depending on the stressed condition. The capability to have more frequent spent media analyses would allow for real-time observation of these process changes and associated control strategies. [ABSTRACT FROM AUTHOR]