To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell-specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time-course profiles of HCP (as measured by multi-analyte ELISA) and those of two intracellular HCP species, phospholipase B-like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC-MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ∼80% to <20% and extracellular LDH levels increased several-fold. Our findings indicate that the CHO-derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (>10(7) cells/mL)-operated in fed-batch mode and exhibiting high viabilities (>70%) throughout the culture duration-can accumulate a considerable amount of immunogenic HCP (∼1-2 g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC-MS/MS to overcome the limitations associated with ELISA and 2D-PAGE for HCP analysis.

During production of therapeutic monoclonal antibodies (mAb), it is highly desirable to remove and control antibody aggregates in the manufacturing process to minimize the potential risk of immunogenicity to patients. During process development for the production of a recombinant IgG in a CHO cell line, we observed atypical high variability from 1 to 20% mAb aggregates formed during cell culture that negatively impacted antibody purification. Analytical characterization revealed the IgG aggregates were mediated by hydrophobic interactions likely caused by misfolded antibody during intracellular processing. Strikingly, data analysis showed an inverse correlation of lower cell culture temperature producing higher aggregate levels. All cultures at 37°C exhibited ≤ 5% aggregates at harvest. Aggregate levels increased 4-12-fold in 33°C cultures when compared to 37°C, with a corresponding 2-4-fold increase in heavy chain (HC) and light chain (LC) mRNA. Additionally, 37°C cases showed a greater excess of LC to HC mRNA levels. Endoplasmic reticulum (ER) chaperone expression and ER size also increased 25-75% at 33°C versus 37°C but to a lesser extent than LC and HC mRNA, consistent with a potential limiting ER folding capacity at 33°C for this cell line. Finally, we identified a 2-5-fold increase in mAb aggregate formation at 33°C compared to 37°C cultures for three additional CHO cell lines. Taken together, our observations indicate that low culture temperature can increase antibody aggregate formation in CHO cells by increasing LC and HC transcripts coupled with limited ER machinery.

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Background: Protein kinase A has been shown to be involved in modulating critical functions. Results: Although the activity of PknA is crucial for cell growth, the extracellular domain is expendable. Conclusion: Although the activation of PknA is necessary for its function, this is independent of PknB. Significance: PknA plays an indispensable role and is required for both in vitro and in vivo growth.

The rapid growth of the world population has increased the food demand as well as the need for assurance of food quality, safety, and sustainability. However, food security can easily be compromised by not only natural hazards but also changes in food preferences, political conflicts, and food frauds. In order to contribute to building a more sustainable food system—digitally visible and processes measurable—within this review, we summarized currently available evidence for various information and communication technologies (ICTs) that can be utilized to support collaborative actions, prevent fraudulent activities, and remotely perform real-time monitoring, which has become essential, especially during the COVID-19 pandemic. The Internet of Everything, 6G, blockchain, artificial intelligence, and digital twin are gaining significant attention in recent years in anticipation of leveraging the creativity of human experts in collaboration with efficient, intelligent, and accurate machines, but with limited consideration in the food supply chain. Therefore, this paper provided a thorough review of the food system by showing how various ICT tools can help sense and quantify the food system and highlighting the key enhancements that Industry 5.0 technologies can bring. The vulnerability of the food system can be effectively mitigated with the utilization of various ICTs depending on not only the nature and severity of crisis but also the specificity of the food supply chain. There are numerous ways of implementing these technologies, and they are continuously evolving.