Using MVDA with stoichiometric balances to optimize amino acid concentrations in chemically defined CHO cell culture medium for improved culture performance.

Chemically defined (CD) media are routinely used in the production of biologics in Chinese hamster ovary (CHO) cell culture and provide enhanced raw material control. Nutrient optimized CD media is an important path to increase cell growth and monoclonal antibody (mAb) productivity in recombinant CHO cell lines. However, nutrient optimization efforts for CD media typically rely on multifactorial and experimental design of experiment approaches or complex mathematical models of cellular metabolism or gene expression systems. Moreover, the majority of these efforts are aimed at amino acids since they constitute essential nutrients in CD media as they directly contribute to biomass and protein production. In this study, we demonstrate the utilization of multivariate data analytics (MVDA) coupled with amino acid stoichiometric balances (SBs) to increased cell growth and mAb productivity in efforts to support CD media development efforts. SBs measure the difference between theoretical demand of amino acids and the empirically measured fluxes to identify various catabolic or anabolic states of the cell. When coupled with MVDA, the statistical models were not only able to highlight key amino acids toward cell growth or productivity, but also provided direction on metabolic favorability of the amino acid. Experimental validation of our approach resulted in a 55% increase in total cell growth and about an 80% increase in total mAb productivity. Increased specific consumption of stoichiometrically balanced amino acids and decreased specific consumption of glucose was also observed in optimized CD media suggesting favorable consumption of desired nutrients and a potential for energy redistribution toward increased cellular growth and mAb productivity.

Multi-omics profiling of a CHO cell culture system unravels the effect of culture pH on cell growth, antibody titer, and product quality.

A robust monoclonal antibody (mAb) bioprocess requires physiological parameters such as temperature, pH, or dissolved oxygen to be well-controlled as even small variations in them could potentially impact the final product quality. For instance, pH substantially affects N-glycosylation, protein aggregation, and charge variant profiles, as well as mAb productivity. However, relatively less is known about how pH jointly influences product quality and titer. In this study, we investigated the effect of pH on culture performance, product titer, and quality profiles by applying longitudinal multi-omics profiling, including transcriptomics, proteomics, metabolomics, and glycomics, at three different culture pH set points. The subsequent systematic analysis of multi-omics data showed that pH set points differentially regulated various intracellular pathways including intracellular vesicular trafficking, cell cycle, and apoptosis, thereby resulting in differences in specific productivity, product titer, and quality profiles. In addition, a time-dependent variation in mAb N-glycosylation profiles, independent of pH, was identified to be mainly due to the accumulation of mAb proteins in the endoplasmic reticulum disrupting cellular homeostasis over culture time. Overall, this multi-omics-based study provides an in-depth understanding of the intracellular processes in mAb-producing CHO cell line under varied pH conditions, and could serve as a baseline for enabling the quality optimization and control of mAb production.

Investigating the Role of TNF-α and IFN-γ Activation on the Dynamics of iNOS Gene Expression in LPS Stimulated Macrophages.

Macrophage produced inducible nitric oxide synthase (iNOS) is known to play a critical role in the proinflammatory response against intracellular pathogens by promoting the generation of bactericidal reactive nitrogen species. Robust and timely production of nitric oxide (NO) by iNOS and analogous production of reactive oxygen species are critical components of an effective immune response. In addition to pathogen associated lipopolysaccharides (LPS), iNOS gene expression is dependent on numerous proinflammatory cytokines in the cellular microenvironment of the macrophage, two of which include interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α). To understand the synergistic effect of IFN-γ and TNF-α activation, and LPS stimulation on iNOS expression dynamics and NO production, we developed a systems biology based mathematical model. Using our model, we investigated the impact of pre-infection cytokine exposure, or priming, on the system. We explored the essentiality of IFN-γ priming to the robustness of initial proinflammatory response with respect to the ability of macrophages to produce reactive species needed for pathogen clearance. Results from our theoretical studies indicated that IFN-γ and subsequent activation of IRF1 are essential in consequential production of iNOS upon LPS stimulation. We showed that IFN-γ priming at low concentrations greatly increases the effector response of macrophages against intracellular pathogens. Ultimately the model demonstrated that although TNF-α contributed towards a more rapid response time, measured as time to reach maximum iNOS production, IFN-γ stimulation was significantly more significant in terms of the maximum expression of iNOS and the concentration of NO produced.

In silico Kinetic Model of iNOS expression in macrophages.

Macrophages are a key component in the host innate response and are major contributors to the proinflammatory response against pathogens. One of the key players in the proinflammatory response is induced nitric oxide synthase (iNOS), an enzyme that provides the nitric oxide needed by phagocytic cells to create reactive nitrogen species, which are highly damaging to intracellular pathogens. To model the macrophage intracellular mechanism of iNOS gene expression, we use a systems biology approach to capture the dynamics of the iNOS gene expression system stimulated by bacterial lipopolysaccharide (LPS) and IFN-γ. Our simulation results agree with in vitro assays of iNOS gene expression and provide a platform for further investigating the potential impact of LPS and IFN-γ variations on macrophage effector function.

Contamination risk of water quality on fisheries and aquatic life of Tigris River reach in Mosul City/Noth Iraq.

Considerable attention is now being paid to the use of rivers and lakes for such purposes as fishing, aquatic life and esthetical enjoyment. A study of the water quality of the Tigris River in Mosul city, North Iraq, was carried out in order to assess the chemical composition and suitability for fisheries and aquatic life. Laboratory analysis of 9 water samples for three sites within the rivers reach were performed in order to obtain the chemical concentration of Ca, Na, Mg, HCO3, SO4, Cl, NO3, NO2, TDS and pH. Certain ions may be lethal to fish and other aquatic lifeforms when present at levels near or below the limit given by public water supplies. Copper, zinc and aluminum, which are not among the metals for which limits are prescribed for public water supply, are toxic to fish and many other species of aquatic life. Concentration of chemical constituents in water samples vary spatially and temporarily. The chemical analysis of the water samples was compared with the standard guideline values as recommended by the World Health Organization (WHO) for fisheries and aquatic life. Three samples from the right bank of the river fell within the poor quality type for water. While the other samples from the left side and the deepest part of the river fell within the high quality type water.