Analytical sameness methodology for the evaluation of structural, physicochemical, and biological characteristics of Armlupeg: A pegfilgrastim biosimilar case study.

Pegfilgrastim is administered as an adjunct to chemotherapy to reduce the incidence of febrile neutropenia and associated infectious complications. Lupin's Pegfilgrastim is a proposed biosimilar to the U.S.-referenced Neulasta®. Demonstration of biosimilarity requires extensive physicochemical and functional characterization of the biosimilar, and demonstration of analytical similarity to the reference product, in addition to clinical studies. This work is a case study for demonstrating the analytical similarity of Armlupeg (Lupin's Pegfilgrastim) to Neulasta® with respect to structural and physicochemical attributes using several robust, orthogonal, and state-of-the-art techniques including high-end liquid chromatography, mass spectrometry, and spectroscopy techniques; circular dichroism; differential scanning calorimetry; nuclear magnetic resonance; analytical ultracentrifugation; and micro-flow imaging. Functional similarity was demonstrated using an in vitro cell proliferation assay to measure relative potency and surface plasmon resonance to measure receptor binding kinetics. Furthermore, comparative forced-degradation studies were performed to study the degradation of the products under stress conditions. The product attributes were ranked based on a critical quality attributes risk score according to their potential clinical impact. Based on criticality, all analyses were statistically evaluated to conclude analytical similarity. Lupin's Pegfilgrastim was comparable to Neulasta® as demonstrated via structural, functional, and purity analyses. Lupin's Pegfilgrastim complied with the quality and statistical ranges established using Neulasta®. Both products follow the same degradation pathways under stress conditions as observed in the forced-degradation studies. No new impurity or degradation product was observed in Lupin's Pegfilgrastim. These data conclusively demonstrate the analytical similarity of Lupin's Pegfilgrastim and Neulasta®.

Proteomic study of endothelial dysfunction induced by AGEs and its possible role in diabetic cardiovascular complications.

Endothelial dysfunction is one of the primary steps in the development of diabetes associated cardiovascular diseases. Hyperglycemic condition in diabetes promotes accumulation of advanced glycation end products (AGEs) in the plasma, that interact with the receptor for AGEs (RAGE) present on the endothelial cells and negatively affect their function. Using Human umbilical vascular endothelial cells (HUVECs) in culture, the effect of glycated human serum albumin on global proteomic changes was studied by SWATH-MS, a label free quantitative proteomic approach. Out of the 1860 proteins identified, 161 showed higher abundance while 123 showed lesser abundance in cells treated with glycated HSA. Bioinformatic analysis revealed that the differentially regulated proteins were involved in various processes such as apoptosis, oxidative stress etc. that are associated with endothelial dysfunction. Furthermore, the iRegulon analysis and immunofuorescence studies indicated that several of the differentially regulated proteins were transcriptionally regulated by NF-κB, that is downstream to AGE-RAGE axis. Some of the important differentially regulated proteins include ICAM1, vWF, PAI-1 that affect important endothelial functions like cell adhesion and blood coagulation. qPCR analysis showed an increase in expression of the AGE receptor RAGE along with other genes involved in endothelial function. AGE treatment to HUVEC cells led to increased oxidative stress and apoptosis. This is the first proteomics study that provides insight into proteomic changes downstream to AGE-RAGE axis leading to endothelial dysfunction and predisposing to cardiovascular complications. SIGNIFICANCE: Cardiovascular disease (CVD) is a major pathological outcome in diabetic patients and it is important to address ways that target its development before the onset. Elevated plasma AGEs in diabetes can affect endothelial function and can continue to show their effects even after blood glucose levels are back to normal. Since endothelial dysfunction acts as one of the initiating factors for the development of CVD, understanding how AGEs affect the endothelial cell proteome to cause dysfunction will provide insight into the mechanisms involved and aid designing new therapeutic approaches.

Methylglyoxal attenuates insulin signaling and downregulates the enzymes involved in cholesterol biosynthesis.

Methylglyoxal (MG) is a highly reactive dicarbonyl known to be elevated under the hyperglycemic conditions of diabetes and is implicated in the development of diabetic complications. Therefore, the current study investigates the role of MG in exacerbating insulin resistance at the insulin signaling level, as well as its effect on the global proteomic level. By using insulin sensitive rat muscle cells (L6) and Chinese hamster ovary (CHO) cells stably expressing the insulin receptor (IR) and a glucose transporter fused with green fluorescent protein (GLUT4-GFP), we have observed that MG impairs insulin signaling, inhibits GLUT4 translocation and reduces glucose uptake. SWATH MS analysis, a label-free quantitative mass spectrometric approach, showed altered expression of 99 proteins out of 2404 identified in response to MG treatment. These proteins are mainly involved in stress response, protein folding and proteolysis. Some of the deregulated proteins such as thioredoxin 2, glutathione S transferase, T complex protein 1 subunit ß (tcbp1), heat shock protein 90 and E3 ubiquitin ligase were previously reported to be associated with either diabetes or insulin resistance. Interestingly, aminoguanidine (AMG), a potent dicarbonyl scavenger, restored the deleterious effects of MG. For the first time, we report that MG induces downregulation of enzymes involved in cholesterol biosynthesis such as acetyl-CoA acetyltransferase, hydroxymethylglutaryl-CoA synthase, farnesyl pyrophosphate synthetase, squalene monooxygenase, and lanosterol synthase. GC MS analysis for sterol metabolites corroborated the proteomic results; MG significantly reduced cholesterol production whereas AMG treatment restored cholesterol production to levels similar to the control. Thus, MG leads to primary defects in insulin signaling and cellular abnormalities at the proteomic and metabolic levels, both of which may contribute to the development of insulin resistance.

Glycation inhibitors extend yeast chronological lifespan by reducing advanced glycation end products and by back regulation of proteins involved in mitochondrial respiration.

Advanced Glycation End products (AGEs) are implicated in aging process. Thus, reducing AGEs by using glycation inhibitors may help in attenuating the aging process. In this study using Saccharomyces cerevisiae yeast system, we show that Aminoguanidine (AMG), a well-known glycation inhibitor, decreases the AGE modification of proteins in non-calorie restriction (NR) (2% glucose) and extends chronological lifespan (CLS) similar to that of calorie restriction (CR) condition (0.5% glucose). Proteomic analysis revealed that AMG back regulates the expression of differentially expressed proteins especially those involved in mitochondrial respiration in NR condition, suggesting that it switches metabolism from fermentation to respiration, mimicking CR. AMG induced back regulation of differentially expressed proteins could be possibly due to its chemical effect or indirectly by glycation inhibition. To delineate this, Metformin (MET), a structural analog of AMG and a mild glycation inhibitor and Hydralazine (HYD), another potent glycation inhibitor but not structural analog of AMG were used. HYD was more effective than MET in mimicking AMG suggesting that glycation inhibition was responsible for restoration of differentially expressed proteins. Thus glycation inhibitors particularly AMG, HYD and MET extend yeast CLS by reducing AGEs, modulating the expression of proteins involved in mitochondrial respiration and possibly by scavenging glucose. SIGNIFICANCE: This study reports the role of glycation in aging process. In the non-caloric restriction condition, carbohydrates such as glucose promote protein glycation and reduce CLS. While, the inhibitors of glycation such as AMG, HYD, MET mimic the caloric restriction condition by back regulating deregulated proteins involved in mitochondrial respiration which could facilitate shift of metabolism from fermentation to respiration and extend yeast CLS. These findings suggest that glycation inhibitors can be potential molecules that can be used in management of aging.

A retrospective analysis of the effect of discussion in teleconference and face-to-face scientific peer-review panels.

OBJECTIVE: With the use of teleconferencing for grant peer-review panels increasing, further studies are necessary to determine the efficacy of the teleconference setting compared to the traditional onsite/face-to-face setting. The objective of this analysis was to examine the effects of discussion, namely changes in application scoring premeeting and postdiscussion, in these settings. We also investigated other parameters, including the magnitude of score shifts and application discussion time in face-to-face and teleconference review settings. DESIGN: The investigation involved a retrospective, quantitative analysis of premeeting and postdiscussion scores and discussion times for teleconference and face-to-face review panels. The analysis included 260 and 212 application score data points and 212 and 171 discussion time data points for the face-to-face and teleconference settings, respectively. RESULTS: The effect of discussion was found to be small, on average, in both settings. However, discussion was found to be important for at least 10% of applications, regardless of setting, with these applications moving over a potential funding line in either direction (fundable to unfundable or vice versa). Small differences were uncovered relating to the effect of discussion between settings, including a decrease in the magnitude of the effect in the teleconference panels as compared to face-to-face. Discussion time (despite teleconferences having shorter discussions) was observed to have little influence on the magnitude of the effect of discussion. Additionally, panel discussion was found to often result in a poorer score (as opposed to an improvement) when compared to reviewer premeeting scores. This was true regardless of setting or assigned reviewer type (primary or secondary reviewer). CONCLUSIONS: Subtle differences were observed between settings, potentially due to reduced engagement in teleconferences. Overall, further research is required on the psychology of decision-making, team performance and persuasion to better elucidate the group dynamics of telephonic and virtual ad-hoc peer-review panels.

Proteome wide reduction in AGE modification in streptozotocin induced diabetic mice by hydralazine mediated transglycation.

The non-enzymatic reaction between glucose and protein can be chemically reversed by transglycation. Here we report the transglycation activity of hydralazine using a newly developed MALDI-TOF-MS based assay. Hydralazine mediated transglycation of HbA1c, plasma proteins and kidney proteins was demonstrated in streptozotocin (STZ) induced diabetic mice, as evidenced by decrease in protein glycation, as well as presence of hydralazine-glucose conjugate in urine of diabetic mice treated with hydralazine. Hydralazine down regulated the expression of Receptor for Advanced Glycation End products (RAGE), NADPH oxidase (NOX), and super oxide dismutase (SOD). These findings will provide a new dimension for developing intervention strategies for the treatment of glycation associated diseases such as diabetes complications, atherosclerosis, and aging.