11 research outputs found
N-Glycan distribution for 5-L treatments.
<p>N-Glycan distribution for 5-L treatments.</p
Proposed biological mechanism for manufacturing and low oxygen laboratory scale bioreactors.
<p>Reduced oxygen levels trigger an oxidative stress response and shift in glucose metabolism, resulting in upregulation of glycolysis and mannose synthesis, and downregulation of the hexosamine pathway and acetyl-CoA formation. This metabolic shift results in reduced GlcNAc levels, as well as levels of metabolites formed from GlcNAc including UDP-GlcNAc and CMP-NANA, triggering a reduction in terminal sialylation of N-glycans. Metabolite or gene expression levels directly measures as increased (green arrows) or decreased (red arrows) are shown. Abbreviations are as follows: CMP, cytidine monophosphate; CMP-SAT, CMP sialic acid transporter; CoA, coenzyme A; CTP, cytidine triphosphate; Frc-6-P, fructose-6-phosphate; Glc-6-P, glucose-6-phosphate; GlcN, glucosamine; GlcNAc, N-acetyl glucosamine; Gln, glutamine; Glu, glutamate; GTP, guanosine triphosphate; Man, mannose; Man-6-P, mannose-6-phosphate; ManNAc, N-acetyl mannosamine; MK, mannosekinase; NANA, N-acetylneuraminic acid; NH<sub>3</sub>, ammonia; PDH, pyruvate dehydrogenase; PDK, pyruvate dehydrogenase kinase; PFK, phosphofructokinase; PMI, mannose phosphate isomerase; UDP, uridine diphosphate; UMP, uridine monophosphate; UTP, uridine triphosphate.</p
N-Glycan Structures.
<p>The N-glycan assay method quantifies the percent distribution of N-glycan species. Five key structures were quantified in samples generated from 5-L bioreactors were operated under normal (50%) and low (15%) DO conditions. G0F, G1F and G2F are unsialylated, S1G1F is monosialylated, and S2G2F is disialylated.</p
Links between glucose metabolism and NANA.
<p>Untargeted metabolomics & glucose consumption calculations were performed on samples ranging from 50-L to 5000-L scale. A. Relative mannose levels, a metabolite not provided in the cell culture media, were compared across runs with normal (red) and low (blue) NANA levels. Error bars represent the mannose range associated with each time point. B. Maximum specific glucose consumption was calculated for each run and found to be significantly inversely correlated to day 10 NANA (p<0.001). C. Residuals of maximum specific glucose consumption were calculated for each run and found to be significantly inversely correlated to day 10 NANA (p<0.0001).</p
Reduced DO level impacts 5-L cell culture gene expression.
<p>5-L bioreactors were operated under control (50% DO, blue) and low DO (20% gray, 15% yellow, and 10% shifted to 20% DO on day 5 green) conditions. Gene expression, relative to the control (50% DO) is shown for oxidative stress (A) and glucose metabolism (B) markers. Replicate bioreactors were used for control (n = 3), and 15% DO (n = 2) conditions. Statistical differences were determined using a student t-test, * indicates p<0.05 and ** indicates p<0.01.</p
Reduced DO level impacts 5-L cell culture performance and sialylation.
<p>5-L bioreactors were operated under control (50% DO, blue circle) and low DO (20% grey triangle, 15% yellow square, 10% red diamond, and 10% shifted to 20% DO on day 5 green diamond) conditions and compared to 5000-L operation (50% DO, orange circle). Viability (A), lactate (B) and ammonia (C) profiles were established for up to 14 days of bioreactor operation. D. Day 10 titer, day 12 NANA and NANA slope values were normalized to the 5-L control (50% DO) condition. Replicate bioreactors were used for control (n = 6), 5000-L (n = 8) and 15% DO (n = 4) conditions. Statistical differences were determined using a student t-test, * indicates p<0.05 and ** indicates p<0.01.</p
Intracellular metabolites impacted by oxygen treatments.
<p>Intracellular metabolites impacted by oxygen treatments.</p
Global microarray targets correlated with NANA level.
<p>Global microarray targets correlated with NANA level.</p
Potent and Selective Agonists of Sphingosine 1‑Phosphate 1 (S1P<sub>1</sub>): Discovery and SAR of a Novel Isoxazole Based Series
Sphingosine 1-phosphate (S1P) is
the endogenous ligand for the
sphingosine 1-phosphate receptors (S1P<sub>1–5</sub>) and evokes
a variety of cellular responses through their stimulation. The interaction
of S1P with the S1P receptors plays a fundamental physiological role
in a number of processes including vascular development and stabilization,
lymphocyte migration, and proliferation. Agonism of S1P<sub>1</sub>, in particular, has been shown to play a significant role in lymphocyte
trafficking from the thymus and secondary lymphoid organs, resulting
in immunosuppression. This article will detail the discovery and SAR
of a potent and selective series of isoxazole based full agonists
of S1P<sub>1</sub>. Isoxazole <b>6d</b> demonstrated impressive
efficacy when administered orally in a rat model of arthritis and
in a mouse experimental autoimmune encephalomyelitis (EAE) model of
multiple sclerosis
Discovery and Structure–Activity Relationship (SAR) of a Series of Ethanolamine-Based Direct-Acting Agonists of Sphingosine-1-phosphate (S1P<sub>1</sub>)
Sphingosine-1-phosphate
(S1P) is a bioactive sphingolipid metabolite
that regulates a multitude of physiological processes such as lymphocyte
trafficking, cardiac function, vascular development, and inflammation.
Because of the ability of S1P<sub>1</sub> receptor agonists to suppress
lymphocyte egress, they have great potential as therapeutic agents
in a variety of autoimmune diseases. In this article, the discovery
of selective, direct acting S1P<sub>1</sub> agonists utilizing an
ethanolamine scaffold containing a terminal carboxylic acid is described.
Potent S1P<sub>1</sub> agonists such as compounds <b>18a</b> and <b>19a</b> which have greater than 1000-fold selectivity
over S1P<sub>3</sub> are described. These compounds efficiently reduce
blood lymphocyte counts in rats through 24 h after single doses of
1 and 0.3 mpk, respectively. Pharmacodynamic properties of both compounds
are discussed. Compound <b>19a</b> was further studied in two
preclinical models of disease, exhibiting good efficacy in both the
rat adjuvant arthritis model (AA) and the mouse experimental autoimmune
encephalomyelitis model (EAE)