Untargeted proteomics reveals upregulation of stress response pathways during CHO-based monoclonal antibody manufacturing process leading to disulfide bond reduction.

Monoclonal antibody (mAb) interchain disulfide bond reduction can cause a loss of function and negatively impact the therapeutic's efficacy and safety. Disulfide bond reduction has been observed at various stages during the manufacturing process, including processing of the harvested material. The factors and mechanisms driving this phenomenon are not fully understood. In this study, we examined the host cell proteome as a potential factor affecting the susceptibility of a mAb to disulfide bond reduction in the harvested cell culture fluid (HCCF). We used untargeted liquid-chromatography-mass spectrometry-based proteomics experiments in conjunction with a semi-automated protein identification workflow to systematically compare Chinese hamster ovary (CHO) cell protein abundances between bioreactor conditions that result in reduction-susceptible and reduction-free HCCF. Although the growth profiles and antibody titers of these two bioreactor conditions were indistinguishable, we observed broad differences in host cell protein (HCP) expression. We found significant differences in the abundance of glycolytic enzymes, key protein reductases, and antioxidant defense enzymes. Multivariate analysis of the proteomics data determined that upregulation of stress-inducible endoplasmic reticulum (ER) and other chaperone proteins is a discriminatory characteristic of reduction-susceptible HCP profiles. Overall, these results suggest that stress response pathways activated during bioreactor culture increase the reduction-susceptibility of HCCF. Consequently, these pathways could be valuable targets for optimizing culture conditions to improve protein quality.

Metabolic understanding of disulfide reduction during monoclonal antibody production.

The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF. KEY POINTS: • Bioreactor conditions resulted in reduction susceptible harvest material. • GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction. • Demonstrated role for cell metabolic changes in post-harvest mAb reduction. Graphical abstract.

Novel Observations From Next-Generation RNA Sequencing of Highly Purified Human Adult and Fetal Islet Cell Subsets.

Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic α- and ß-cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase ß-cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify α-, ß-, and δ-cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the subpopulations by flow cytometry, and, using next-generation RNA sequencing, we report the detailed transcriptomes of fetal and adult α- and ß-cells. We observed that human islet composition was not influenced by age, sex, or BMI, and transcripts for inflammatory gene products were noted in fetal ß-cells. In addition, within highly purified adult glucagon-expressing α-cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet α- and ß-cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes.

Caffeine inhibits glucose transport by binding at the GLUT1 nucleotide-binding site.

Glucose transporter 1 (GLUT1) is the primary glucose transport protein of the cardiovascular system and astroglia. A recent study proposes that caffeine uncompetitive inhibition of GLUT1 results from interactions at an exofacial GLUT1 site. Intracellular ATP is also an uncompetitive GLUT1 inhibitor and shares structural similarities with caffeine, suggesting that caffeine acts at the previously characterized endofacial GLUT1 nucleotide-binding site. We tested this by confirming that caffeine uncompetitively inhibits GLUT1-mediated 3-O-methylglucose uptake in human erythrocytes [Vmax and Km for transport are reduced fourfold; Ki(app) = 3.5 mM caffeine]. ATP and AMP antagonize caffeine inhibition of 3-O-methylglucose uptake in erythrocyte ghosts by increasing Ki(app) for caffeine inhibition of transport from 0.9 ± 0.3 mM in the absence of intracellular nucleotides to 2.6 ± 0.6 and 2.4 ± 0.5 mM in the presence of 5 mM intracellular ATP or AMP, respectively. Extracellular ATP has no effect on sugar uptake or its inhibition by caffeine. Caffeine and ATP displace the fluorescent ATP derivative, trinitrophenyl-ATP, from the GLUT1 nucleotide-binding site, but d-glucose and the transport inhibitor cytochalasin B do not. Caffeine, but not ATP, inhibits cytochalasin B binding to GLUT1. Like ATP, caffeine renders the GLUT1 carboxy-terminus less accessible to peptide-directed antibodies, but cytochalasin B and d-glucose do not. These results suggest that the caffeine-binding site bridges two nonoverlapping GLUT1 endofacial sites-the regulatory, nucleotide-binding site and the cytochalasin B-binding site. Caffeine binding to GLUT1 mimics the action of ATP but not cytochalasin B on sugar transport. Molecular docking studies support this hypothesis.

The pancreatic ß-cell transcriptome and integrated-omics.

PURPOSE OF REVIEW: ß Cells represent one of many cell types in heterogeneous pancreatic islets and play the central role in maintaining glucose homeostasis, such that disrupting ß-cell function leads to diabetes. This review summarizes the methods for isolating and characterizing ß cells, and describes integrated 'omics' approaches used to define the ß cell by its transcriptome and proteome. RECENT FINDINGS: RNA sequencing and mass spectrometry-based protein identification have now identified RNA and protein profiles for mouse and human pancreatic islets and ß cells, and for ß-cell lines. Recent publications have outlined these profiles and, more importantly, have begun to assign the presence or absence of specific genes and regulatory molecules to ß-cell function and dysfunction. Overall, researchers have focused on understanding the pathophysiology of diabetes by connecting genome, transcriptome, proteome, and regulatory RNA profiles with findings from genome-wide association studies. SUMMARY: Studies employing these relatively new techniques promise to identify specific genes or regulatory RNAs with altered expression as ß-cell function begins to deteriorate in the spiral toward the development of diabetes. The ultimate goal is to identify the potential therapeutic targets to prevent ß-cell dysfunction and thereby better treat the individual with diabetes. VIDEO ABSTRACT: http://links.lww.com/COE/A5.

Role of monosaccharide transport proteins in carbohydrate assimilation, distribution, metabolism, and homeostasis.

The facilitated diffusion of glucose, galactose, fructose, urate, myoinositol, and dehydroascorbicacid in mammals is catalyzed by a family of 14 monosaccharide transport proteins called GLUTs. These transporters may be divided into three classes according to sequence similarity and function/substrate specificity. GLUT1 appears to be highly expressed in glycolytically active cells and has been coopted in vitamin C auxotrophs to maintain the redox state of the blood through transport of dehydroascorbate. Several GLUTs are definitive glucose/galactose transporters, GLUT2 and GLUT5 are physiologically important fructose transporters, GLUT9 appears to be a urate transporter while GLUT13 is a proton/myoinositol cotransporter. The physiologic substrates of some GLUTs remain to be established. The GLUTs are expressed in a tissue specific manner where affinity, specificity, and capacity for substrate transport are paramount for tissue function. Although great strides have been made in characterizing GLUT-catalyzed monosaccharide transport and mapping GLUT membrane topography and determinants of substrate specificity, a unifying model for GLUT structure and function remains elusive. The GLUTs play a major role in carbohydrate homeostasis and the redistribution of sugar-derived carbons among the various organ systems. This is accomplished through a multiplicity of GLUT-dependent glucose sensing and effector mechanisms that regulate monosaccharide ingestion, absorption,distribution, cellular transport and metabolism, and recovery/retention. Glucose transport and metabolism have coevolved in mammals to support cerebral glucose utilization.

AMP kinase regulation of sugar transport in brain capillary endothelial cells during acute metabolic stress.

AMP-dependent kinase (AMPK) and GLUT1-mediated sugar transport in blood-brain barrier endothelial cells are activated during acute cellular metabolic stress. Using murine brain microvasculature endothelium bEnd.3 cells, we show that AMPK phosphorylation and stimulation of 3-O-methylglucose transport by the AMPK agonist AICAR are inhibited in a dose-dependent manner by the AMPK antagonist Compound C. AMPK α1- or AMPK α2-knockdown by RNA interference or AMPK inhibition by Compound C reduces AMPK phosphorylation and 3-O-methylglucose transport stimulation induced by cellular glucose-depletion, by potassium cyanide (KCN), or by carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (FCCP). Cell surface biotinylation studies reveal that plasma membrane GLUT1 levels are increased two- to threefold by cellular glucose depletion, AICAR or KCN treatment, and that these increases are prevented by Compound C and by AMPK α1- or α2-knockdown. These results support the hypothesis that AMPK activation in blood-brain barrier-derived endothelial cells directs the trafficking of GLUT1 intracellular pools to the plasma membrane, thereby increasing endothelial sugar transport capacity.

Acute modulation of sugar transport in brain capillary endothelial cell cultures during activation of the metabolic stress pathway.

GLUT1-catalyzed equilibrative sugar transport across the mammalian blood-brain barrier is stimulated during acute and chronic metabolic stress; however, the mechanism of acute transport regulation is unknown. We have examined acute sugar transport regulation in the murine brain microvasculature endothelial cell line bEnd.3. Acute cellular metabolic stress was induced by glucose depletion, by potassium cyanide, or by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which reduce or deplete intracellular ATP within 15 min. This results in a 1.7-7-fold increase in V(max) for zero-trans 3-O-methylglucose uptake (sugar uptake into sugar-free cells) and a 3-10-fold increase in V(max) for equilibrium exchange transport (intracellular [sugar] = extracellular [sugar]). GLUT1, GLUT8, and GLUT9 mRNAs are detected in bEnd.3 cells where GLUT1 mRNA levels are 33-fold greater than levels of GLUT8 or GLUT9 mRNA. Neither GLUT1 mRNA nor total protein levels are affected by acute metabolic stress. Cell surface biotinylation reveals that plasma membrane GLUT1 levels are increased 2-3-fold by metabolic depletion, although cell surface Na(+),K(+)-ATPase levels remain unaffected by ATP depletion. Treatment with the AMP-activated kinase agonist, AICAR, increases V(max) for net 3-O-methylglucose uptake by 2-fold. Glucose depletion and treatment with potassium cyanide, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and AICAR also increase AMP-dependent kinase phosphorylation in bEnd.3 cells. These results suggest that metabolic stress rapidly stimulates blood-brain barrier endothelial cell sugar transport by acute up-regulation of plasma membrane GLUT1 levels, possibly involving AMP-activated kinase activity.

Dolichol-linked oligosaccharide selection by the oligosaccharyltransferase in protist and fungal organisms.

The dolichol-linked oligosaccharide Glc3Man9GlcNAc2-PP-Dol is the in vivo donor substrate synthesized by most eukaryotes for asparagine-linked glycosylation. However, many protist organisms assemble dolichol-linked oligosaccharides that lack glucose residues. We have compared donor substrate utilization by the oligosaccharyltransferase (OST) from Trypanosoma cruzi, Entamoeba histolytica, Trichomonas vaginalis, Cryptococcus neoformans, and Saccharomyces cerevisiae using structurally homogeneous dolichol-linked oligosaccharides as well as a heterogeneous dolichol-linked oligosaccharide library. Our results demonstrate that the OST from diverse organisms utilizes the in vivo oligo saccharide donor in preference to certain larger and/or smaller oligosaccharide donors. Steady-state enzyme kinetic experiments reveal that the binding affinity of the tripeptide acceptor for the protist OST complex is influenced by the structure of the oligosaccharide donor. This rudimentary donor substrate selection mechanism has been refined in fungi and vertebrate organisms by the addition of a second, regulatory dolichol-linked oligosaccharide binding site, the presence of which correlates with acquisition of the SWP1/ribophorin II subunit of the OST complex.