Natural products are excellent sources of lead compounds in the search for novel drugs for treatment of various diseases. The most under-explored source of such materials lies in the tropical and subtropical regions of the world. In these areas, a long tradition of ethnobotanical medicine often exists and offers a rich and relatively untapped source for the discovery of novel drugs from natural products. 1) A traditional Vietnamese medicinal plant, Tetracera scandens is originally from the Quang Ninh province in Vietnam. It is called 'Day Chieu' in Vietnamese and has shown some therapeutic activities in inflammation, hepatitis and gout. 1) However, no reports on the effects of T. scandens and its components have been published in the field of diabetes.Type 2 diabetes mellitus (T2DM) is a heterogeneous metabolic disorder characterized by the impairment of insulin-secretion from pancreatic beta cells and insulin resistance in peripheral tissues such as liver, adipose tissue, and skeletal muscle. 2) Skeletal muscles account for approximately 75% of glucose absorption under insulin-stimulated conditions, and a reduction in insulin-stimulated glucose-uptake in skeletal muscles of T2DM patients has been observed both in vitro and in vivo. 3,4) Some commonly used glucose-lowering antidiabetic drugs in the market such as metformin, rosiglitazone and pioglitazone are believed to increase the glucose-uptake in skeletal muscle. 5,6) Therefore, muscle glucose-uptake could be considered as an excellent target for treatment of T2DM.Adenosine monophosphate-activated protein kinase (AMPK) plays a central role in the regulation of glucose and lipid metabolism as an intracellular energy sensor. Upon activation by allosteric binding of AMP or phosphorylation at Thr 172 of its catalytic subunit, AMPK accelerates ATP-generating catabolic pathways, including glucose-uptake and glucose and fatty acid oxidation. 7) In many reports, AMPK-activators such as aminoimidazole carboxamide ribonucleotide (AICAR), peroxisome proliferator-activated receptor g (PPARg) agonists, metformin and berberine stimulated muscle glucose-uptake both in cells and in humans. [8][9][10] Moreover, the uptake of glucose into tissues is mediated to a large extent by the members of a series of facilitated carrier proteins, designated glucose transport protein (GLUT) 1-12. 11) GLUT4 is highly expressed in skeletal muscle cells that exhibit regulated insulin-responsive glucose-uptake. The increased expression of GLUT4 has previously been lowered blood glucose and enhanced glucose transport and glucose utilization in skeletal muscles. 12) GLUT1 is nearly ubiquitous in its distribution and is thought to be primarily responsible for basal glucose-transport associated with AMPK activation. 13,14) The over-expression of GLUT1 in L6 myoblasts and transgenic mice resulted in augmented basal rates of glucose-uptake. 15,16) Thus, basal and insulin-stimulated glucoseuptake into skeletal muscle may be regulated at least in part by the level of GLUT1 and GLUT4 expressions in mu...

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Mammalian cells consume large amount of nutrients during growth and production. However, endogenous metabolic inefficiencies often prevent cells to fully utilize nutrients to support growth and protein production. Instead, significant fraction of fed nutrients is diverted into extracellular accumulation of waste by-products and metabolites, further inhibiting proliferation and protein synthesis. In this study, an LC-MS/MS based metabolomics pipeline was used to screen Chinese hamster ovary (CHO) extracellular metabolites. Six out of eight identified inhibitory metabolites, caused by the inefficient cell metabolism, were not previously studied in CHO cells: aconitic acid, 2-hydroxyisocaproic acid, methylsuccinic acid, cytidine monophosphate, trigonelline, and n-acetyl putrescine. When supplemented back into a fed-batch culture, significant reduction in cellular growth was observed in the presence of each metabolite and all the identified metabolites were shown to impact the glycosylation of a model secreted antibody, with seven of these also reducing CHO cellular productivity (titer) and all eight inhibiting the formation of mono-galactosylated biantennary (G1F) and biantennary galactosylated (G2F) N-glycans. These inhibitory metabolites further impact the metabolism of cells, leading to a significant reduction in CHO cellular growth and specific productivity in fed-batch culture (maximum reductions of 27.2% and 40.6% respectively). In-depth pathway analysis revealed that these metabolites are produced when cells utilize major energy sources such as glucose and select amino acids (tryptophan, arginine, isoleucine, and leucine) for growth, maintenance, and protein production. Furthermore, these novel inhibitory metabolites were observed to accumulate in multiple CHO cell lines (CHO–K1 and CHO-GS) as well as HEK293 cell line. This study provides a robust and holistic methodology to incorporate global metabolomic analysis into cell culture studies for elucidation and structural verification of novel metabolites that participate in key metabolic pathways to growth, production, and post-translational modification in biopharmaceutical production.

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Process intensification of monoclonal antibody production is leading to more concentrated feed media causing issues with precipitation of solids from the media solution. This results in processing problems since components in the precipitate are no longer in solution, changing the media composition and leading to variability in cell culture performance. The goal of this work is to characterize the feed media precipitate, and in particular to identify the precipitated components so that mitigation strategies can be developed. From the conducted analysis, the precipitate was predominately found to be organic and was analyzed with liquid chromatography‐mass spectrometry and inductively coupled plasma‐optical emission spectroscopy (ICP‐OES) to identify the constituent components. Up to ten amino acids were identified with tyrosine (approximately 77 wt.%) and phenylalanine (approximately 4 wt.%) being the most prevalent amino acids. Elemental analysis with ICP‐OES revealed that inorganic components were accounted for less than one weight percentage of the solid precipitate with metal sulfates being the predominant inorganic components.

Previously, we identified six inhibitory metabolites (IMs) accumulating in Chinese hamster ovary (CHO) cultures using AMBIC 1.0 community reference medium that negatively impacted culture performance. The goal of the current study was to modify the medium to control IM accumulation through design of experiments (DOE). Initial over‐supplementation of precursor amino acids (AAs) by 100% to 200% in the culture medium revealed positive correlations between initial AA concentrations and IM levels. A screening design identified 5 AA targets, Lys, Ile, Trp, Leu, Arg, as key contributors to IMs. Response surface design analysis was used to reduce initial AA levels between 13% and 33%, and these were then evaluated in batch and fed‐batch cultures. Lowering AAs in basal and feed medium and reducing feed rate from 10% to 5% reduced inhibitory metabolites HICA and NAP by up to 50%, MSA by 30%, and CMP by 15%. These reductions were accompanied by a 13% to 40% improvement in peak viable cell densities and 7% to 50% enhancement in IgG production in batch and fed‐batch processes, respectively. This study demonstrates the value of tuning specific AA levels in reference basal and feed media using statistical design methodologies to lower problematic IMs.

Therapeutic protein productivity and glycosylation pattern highly rely on cell metabolism. Cell culture medium composition and feeding strategy are critical to regulate cell metabolism. In this study, the relationship between toxic metabolic inhibitors and their nutrient precursors was explored to identify the critical medium components toward cell growth and generation of metabolic by‐products. Generic CHO metabolic model was tailored and integrated with CHO fed‐batch metabolomic data to obtain a cell line‐ and process‐specific model. Flux balance analysis study was conducted on toxic metabolites cytidine monophosphate, guanosine monophosphate and n‐acetylputrescine—all of which were previously reported to generate from endogenous cell metabolism—by mapping them to a compartmentalized carbon utilization network. Using this approach, the study projected high level of inhibitory metabolites accumulation when comparing three industrially relevant fed‐batch feeding conditions one against another, from which the results were validated via a dose‐dependent amino acids spiking study. In the end, a medium optimization design was employed to lower the amount of supplemented nutrients, of which improvements in critical process performance were realized at 40% increase in peak viable cell density (VCD), 15% increase in integral VCD, and 37% increase in growth rate. Tight control of toxic by‐products was also achieved, as the study measured decreased inhibitory metabolites accumulation across all conditions. Overall, the study successfully presented a digital twin approach to investigate the intertwined relationship between supplemented medium constituents and downstream toxic metabolites generated through host cell metabolism, further elucidating different control strategies capable of improving cellular phenotypes and regulating toxic inhibitors.