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N-lined glycosylation is one of the critical quality attributes (CQA) for biotherapeutics impacting the safety and activity of drug product. Changes in pattern and level of glycosylation can significantly alter the intrinsic properties of the product and, therefore, have to be monitored throughout its lifecycle. Therefore fast, precise, and unbiased N-glycan mapping assay is desired. To ensure these qualities, using analytical methods that evaluate completeness of deglycosylation is necessary. For quantification of deglycosylation yield, methods such as reduced liquid chromatography–mass spectrometry (LC-MS) and reduced capillary gel electrophoresis (CGE) have been commonly used. Here we present development of two additional methods to evaluate deglycosylation yield: one based on LC using reverse phase (RP) column and one based on reduced sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE gel) with offline software (GelAnalyzer). With the advent of rapid deglycosylation workflows in the market for N-glycan profiling replacing overnight incubation, we have aimed to quantify the level of deglycosylation in a selected rapid deglycosylation workflow. Our results have shown well resolved peaks of glycosylated and deglycosylated protein species with RP-LC method allowing simple quantification of deglycosylation yield of protein with high confidence. Additionally a good correlation, ≥0.94, was found between deglycosylation yields estimated by RP-LC method and that of reduced SDS-PAGE gel method with offline software. Evaluation of rapid deglycosylation protocol from GlycanAssure™ HyPerformance assay kit performed on fetuin and RNase B has shown complete deglycosylation within the recommended protocol time when evaluated with these techniques. Using this kit, N-glycans from NIST mAb were prepared in 1.4 hr and analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh performance LC (UHPLC) equipped with a fluorescence detector (FLD). 37 peaks were resolved with good resolution. Excellent sample preparation repeatability was found with relative standard deviation (RSD) of 0.5% relative area.
Anahita D. Eckard; David R. Dupont; Johnie K. Young. Development of Two Analytical Methods Based on Reverse Phase Chromatographic and SDS-PAGE Gel for Assessment of Deglycosylation Yield in N-Glycan Mapping. BioMed Research International 2018, 2018, 1 -10.
AMA StyleAnahita D. Eckard, David R. Dupont, Johnie K. Young. Development of Two Analytical Methods Based on Reverse Phase Chromatographic and SDS-PAGE Gel for Assessment of Deglycosylation Yield in N-Glycan Mapping. BioMed Research International. 2018; 2018 ():1-10.
Chicago/Turabian StyleAnahita D. Eckard; David R. Dupont; Johnie K. Young. 2018. "Development of Two Analytical Methods Based on Reverse Phase Chromatographic and SDS-PAGE Gel for Assessment of Deglycosylation Yield in N-Glycan Mapping." BioMed Research International 2018, no. : 1-10.
Enzymatic hydrolysis of lignocellulosic biomass is limited by rapid cellulase deactivation, consequently requiring large amounts of enzyme to maintain acceptable biomass conversion. In this study, a new approach to improve lignocellulose hydrolysis was investigated. Performing enzymatic hydrolysis of corn stover (CS) in the presence of polymeric–surfactant micelles (PMs) was demonstrated to improve hydrolysis yield to a greater extent than using only surfactant micelles. Application of 2 % (w/w) of polyethylene glycol (PEG 6000) with casein, Tween-20, and Triton X-100 at levels above the critical micelle concentrations increased the hydrolysis yield of CS containing high-bound lignin (extrusion-pretreated) by up to 87.8, 11.7, and 7.5 %, respectively. These PMs were not effective during enzymatic hydrolysis of biomass lacking lignin (Avicel) or alkali-pretreated CS (7.2 % lignin). The main reasons for the enhanced cellulase activity observed due to PEG-casein, PEG-Tween, and PEG-Triton were enhanced cellulase solubilization; reformation of α-helix substructure; and combination of induced cellulase solubilization, α-helix reformation, and chemical changes in the microstructure of biomass, respectively. Deformation of the cellulase substructure during hydrolysis of biomass and its subsequent reformation in the presence of surfactants were shown in this study for the first time. Chemical changes in the microstructure of biomass (e.g., lignin side changes, C–O bonds, and amorphous cellulose) were found to be another potential reason for the effectiveness of surfactants when they are incubated at above 6 g/L for 72 h with biomass.
Anahita Dehkhoda Eckard; Kasiviswanathan Muthukumarappan; William R Gibbons. The Role of Polymeric Micelles on Chemical Changes of Pretreated Corn Stover, Cellulase Structure, and Adsorption. BioEnergy Research 2013, 7, 389 -407.
AMA StyleAnahita Dehkhoda Eckard, Kasiviswanathan Muthukumarappan, William R Gibbons. The Role of Polymeric Micelles on Chemical Changes of Pretreated Corn Stover, Cellulase Structure, and Adsorption. BioEnergy Research. 2013; 7 (1):389-407.
Chicago/Turabian StyleAnahita Dehkhoda Eckard; Kasiviswanathan Muthukumarappan; William R Gibbons. 2013. "The Role of Polymeric Micelles on Chemical Changes of Pretreated Corn Stover, Cellulase Structure, and Adsorption." BioEnergy Research 7, no. 1: 389-407.
One of the concerns for economical production of ethanol from biomass is the large volume and high cost of the cellulolytic enzymes used to convert biomass into fermentable sugars. The presence of acetyl groups in hemicellulose and lignin in plant cell walls reduces accessibility of biomass to the enzymes and makes conversion a slow process. In addition to low enzyme accessibility, a rapid deactivation of cellulases during biomass hydrolysis can be another factor contributing to the low sugar recovery. As of now, the economical reduction in lignin content of the biomass is considered a bottleneck, and raises issues for several reasons. The presence of lignin in biomass reduces the swelling of cellulose fibrils and accessibility of enzyme to carbohydrate polymers. It also causes an irreversible adsorption of the cellulolytic enzymes that prevents effective enzyme activity and recycling. Amphiphiles, such as surfactants and proteins have been found to improve enzyme activity by several mechanisms of action that are not yet fully understood. Reduction in irreversible adsorption of enzyme to non-specific sites, reduction in viscosity of liquid and surface tension and consequently reduced contact of enzyme with air-liquid interface, and modifications in biomass chemical structure are some of the benefits derived from surface active molecules. Application of some of these amphiphiles could potentially reduce the capital and operating costs of bioethanol production by reducing fermentation time and the amount of enzyme used for saccharification of biomass. In this review article, the benefit of applying amphiphiles at various stages of ethanol production (i.e., pretreatment, hydrolysis and hydrolysis-fermentation) is reviewed and the proposed mechanisms of actions are described.
Anahita Dehkhoda Eckard; Kasiviswanathan Muthukumarappan; William R Gibbons. A Review of the Role of Amphiphiles in Biomass to Ethanol Conversion. Applied Sciences 2013, 3, 396 -419.
AMA StyleAnahita Dehkhoda Eckard, Kasiviswanathan Muthukumarappan, William R Gibbons. A Review of the Role of Amphiphiles in Biomass to Ethanol Conversion. Applied Sciences. 2013; 3 (2):396-419.
Chicago/Turabian StyleAnahita Dehkhoda Eckard; Kasiviswanathan Muthukumarappan; William R Gibbons. 2013. "A Review of the Role of Amphiphiles in Biomass to Ethanol Conversion." Applied Sciences 3, no. 2: 396-419.