scholarly journals Glycan Profile Analysis of Engineered Trastuzumab with Rationally Added Glycosylation Sequons Presents Significantly Increased Glycan Complexity

Pharmaceutics ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 1747
Author(s):  
Esteban Cruz ◽  
Vicki Sifniotis ◽  
Zeynep Sumer-Bayraktar ◽  
Mouhamad Reslan ◽  
Lorna Wilkinson-White ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Profile Analysis ◽  
Physical Stability ◽  
Structural Heterogeneity ◽  
Pharmaceutical Product ◽  
Glycosylation Site ◽  
Glycan Profile ◽  
Common Strategy ◽  
Accelerated Studies ◽  
Self Association

Protein aggregation constitutes a recurring complication in the manufacture and clinical use of therapeutic monoclonal antibodies (mAb) and mAb derivatives. Antibody aggregates can reduce production yield, cause immunogenic reactions, decrease the shelf-life of the pharmaceutical product and impair the capacity of the antibody monomer to bind to its cognate antigen. A common strategy to tackle protein aggregation involves the identification of surface-exposed aggregation-prone regions (APR) for replacement through protein engineering. It was shown that the insertion of N-glycosylation sequons on amino acids proximal to an aggregation-prone region can increase the physical stability of the protein by shielding the APR, thus preventing self-association of antibody monomers. We recently implemented this approach in the Fab region of full-size adalimumab and demonstrated that the thermodynamic stability of the Fab domain increases upon N-glycosite addition. Previous experimental data reported for this technique have lacked appropriate confirmation of glycan occupancy and structural characterization of the ensuing glycan profile. Herein, we mutated previously identified candidate positions on the Fab domain of Trastuzumab and employed tandem mass spectrometry to confirm attachment and obtain a detailed N-glycosylation profile of the mutants. The Trastuzumab glycomutants displayed a glycan profile with significantly higher structural heterogeneity compared to the HEK Trastuzumab antibody, which contains a single N-glycosylation site per heavy chain located in the CH2 domain of the Fc region. These findings suggest that Fab N-glycosites have higher accessibility to enzymes responsible for glycan maturation. Further, we have studied effects on additional glycosylation on protein stability via accelerated studies by following protein folding and aggregation propensities and observed that additional glycosylation indeed enhances physical stability and prevent protein aggregation. Our findings shed light into mAb glycobiology and potential implications in the application of this technique for the development of “biobetter” antibodies.

scholarly journals Physical Stability Comparisons of IgG1-Fc Variants: Effects of N-Glycosylation Site Occupancy and Asp/Gln Residues at Site Asn 297

2014 ◽  
Vol 103 (6) ◽  
pp. 1613-1627 ◽  
Author(s):  
Mohammad A. Alsenaidy ◽  
Solomon Z. Okbazghi ◽  
Jae Hyun Kim ◽  
Sangeeta B. Joshi ◽  
C. Russell Middaugh ◽  
...  
Keyword(s):  
Physical Stability ◽  
Site Occupancy ◽  
Glycosylation Site ◽  
Glycosylation Site Occupancy

scholarly journals Can Short Peptides Be Inhibitors of Serum Amyloid a Protein Aggregation?

Proceedings ◽  
2019 ◽  
Vol 22 (1) ◽  
pp. 38
Author(s):  
Skibiszewska ◽  
Jankowska
Keyword(s):  
Protein Aggregation ◽  
Serum Amyloid A ◽  
Serum Amyloid ◽  
Short Peptides ◽  
Amyloid A ◽  
Serum Amyloid A Protein ◽  
Amyloidogenic Protein ◽  
Protein Molecules ◽  
Self Association

One of the approaches in the design of anti-amyloid drugs is to find compounds that are able to hamper self-association of amyloidogenic protein molecules. [...]

scholarly journals Development of O/W emulsions containing Euterpe oleracea extract and evaluation of photoprotective efficacy

2014 ◽  
Vol 50 (3) ◽  
pp. 639-652 ◽  
Author(s):  
Cláudia Cecilio Daher ◽  
Ipojucan Silva Fontes ◽  
Rayllan de Oliveira Rodrigues ◽  
Gabriel Azevedo de Brito Damasceno ◽  
Daiane dos Santos Soares ◽  
...  
Keyword(s):  
Physical Stability ◽  
Antioxidant Properties ◽  
Polymeric Surfactant ◽  
Palm Tree ◽  
Sodium Polyacrylate ◽  
Euterpe Oleracea ◽  
Protection Factor ◽  
Accelerated Studies ◽  
The Stability

Euterpe oleraceaMart. is a palm tree popularly known as açai, which is primarily found in northern Brazil. The açai's fruits contain anthocyanins, a class of polyphenols to which antioxidant properties have been attributed. The aim of this work was to develop O/W sunscreens emulsions containing açai glycolic extract (AGE) and to evaluate both their physical stability and photoprotective efficacy. Emulsions containing AGE and sunscreens were formulated using different types and concentrations of polymeric surfactant (acrylates/C 10-30 alkyl acrylate crosspolymer and sodium polyacrylate). The influence of two rheology modifiers (polyacrylamide (and) C13-14/isoparaffin (and) Laureth-7 and Carbomer) on the stability was also investigated. Physical stability was evaluated by preliminary and accelerated studies. Emulsions with 1.0% sodium polyacrylate were stable and exhibited non-newtonian pseudoplastic behavior and thixotropy. Photoprotective efficacy was evaluated by in vivo Sun Protection Factor (SPF) and determination of Protection Factor of UVA (PF-UVA). When AGE was added to the sunscreen emulsion, no significant increase in the in vivo SPF value was observed. The emulsion containing AGE showed PF-UVA = 14.97, 1.69 of the SPF/PF-UVA ratio and a critical wavelength value of 378 nm, and may therefore be considered a sunscreen with UVA and UVB protection.

scholarly journals Functional Tea-Infused Set Yoghurt Development by Evaluation of Sensory Quality and Textural Properties

Foods ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1848
Author(s):  
Katarzyna Świąder ◽  
Anna Florowska ◽  
Zuzanna Konisiewicz ◽  
Yen-Po Chen
Keyword(s):  
Sensory Quality ◽  
Profile Analysis ◽  
Physical Stability ◽  
Principal Component ◽  
Textural Properties ◽  
Sensory Profile ◽  
Oolong Tea ◽  
Willingness To Buy ◽  
Quantitative Descriptive

In the present study, the potential to design natural tea-infused set yoghurt was investigated. Three types of tea (Camellia sinensis): black, green and oolong tea as well as lemon balm (Melissa officinalis L.) were used to produce set yoghurt. The sensory quality (using Quantitative Descriptive Profile analysis and consumer hedonic test) and texture analysis, yield stress, physical stability and colour analysis were assessed to describe the profile of the yoghurt and influence of quality attributes of the product on the consumer acceptability of infused yoghurts in comparison with plain yoghurt. Among the analyzed plant additives for yoghurt, addition of 2% oolong tea to the yoghurt allows a functional food to be obtained with satisfactory texture and sensory properties, accepted by consumers at the same level as for control yoghurt. Both types of yoghurt were also characterised by high consumer willingness to buy, which confirms the legitimacy of using oolong tea as a natural, functional yoghurt additive that improves the sensory quality of the product. The high overall quality of yoghurt with oolong tea in comparison to other plant extracts was associated with the intensive peach flavour and odour, nectar and sweet odour and flavour, and the highest creaminess and thickness. That was confirmed by principal component analysis (PCA) where the overall sensory quality of yoghurts was mainly positively correlated with peach flavour and odour, sweet odour and yoghurt odour, while it was negatively correlated with herbs flavor and odour, and green tea flavour and odour. The sensory profile confirmed no differences in textural profile between plain yoghurt and the tea-infused one measured in the mouth, which corresponds to the result of textural properties such as firmness and adhesiveness.

scholarly journals Musashi-1: An Example of How Polyalanine Tracts Contribute to Self-Association in the Intrinsically Disordered Regions of RNA-Binding Proteins

2020 ◽  
Vol 21 (7) ◽  
pp. 2289 ◽  
Author(s):  
Tsai-Chen Chen ◽  
Jie-rong Huang
Keyword(s):  
Nmr Spectroscopy ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Structural Heterogeneity ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Self Association ◽  
As Stress ◽  
Disordered Regions

RNA-binding proteins (RBPs) have intrinsically disordered regions (IDRs) whose biophysical properties have yet to be explored to the same extent as those of the folded RNA interacting domains. These IDRs are essential to the formation of biomolecular condensates, such as stress and RNA granules, but dysregulated assembly can be pathological. Because of their structural heterogeneity, IDRs are best studied by NMR spectroscopy. In this study, we used NMR spectroscopy to investigate the structural propensity and self-association of the IDR of the RBP Musashi-1. We identified two transient α-helical regions (residues ~208–218 and ~270–284 in the IDR, the latter with a polyalanine tract). Strong NMR line broadening in these regions and circular dichroism and micrography data suggest that the two α-helical elements and the hydrophobic residues in between may contribute to the formation of oligomers found in stress granules and implicated in Alzheimer’s disease. Bioinformatics analysis suggests that polyalanine stretches in the IDRs of RBPs may have evolved to promote RBP assembly.

scholarly journals N-Glycan profile analysis of transferrin using a microfluidic compact disc and MALDI-MS

2016 ◽  
Vol 408 (17) ◽  
pp. 4765-4776 ◽  
Author(s):  
Alessandro Quaranta ◽  
Anna Sroka-Bartnicka ◽  
Erik Tengstrand ◽  
Gunnar Thorsén
Keyword(s):  
Profile Analysis ◽  
Compact Disc ◽  
Maldi Ms ◽  
Glycan Profile

scholarly journals High-throughput and site-specific N-glycosylation analysis of human alpha-1-acid glycoprotein offers a great potential for new biomarker discovery

2020 ◽  
pp. mcp.RA120.002433
Author(s):  
Toma Keser ◽  
Marko Tijardović ◽  
Ivan Gornik ◽  
Edita Lukic ◽  
Gordan Lauc ◽  
...  
Keyword(s):  
Type 2 Diabetes ◽  
High Throughput ◽  
Biomarker Discovery ◽  
Solid Phase ◽  
Glycosylation Site ◽  
Site Specific ◽  
Acid Glycoprotein ◽  
Glycan Profile ◽  
Increased Risk

Alpha-1-acid glycoprotein (AGP) is an acute phase glycoprotein in blood, which is primarily synthetized in the liver and whose biological role is not completely understood. It consists of 45% carbohydrates that are present in the form of five N-linked complex glycans. AGP N-glycosylation was shown to be changed in many different diseases and some changes appear to be disease-specific, thus it has a great diagnostic and prognostic potential. However, AGP glycosylation was mainly analyzed in small cohorts and without detailed site-specific glycan information. Here, we developed a cost-effective method for a high-throughput and site-specific N-glycosylation LC-MS analysis of AGP which can be applied on large cohorts, aid in search for novel disease biomarkers and enable better understanding of AGP’s role and function in health and disease. The method does not require isolation of AGP with antibodies and affinity chromatography, but AGP is enriched by acid precipitation from 5 μl of bloodplasma in a 96 well format. After trypsinization, AGP glycopeptides are purified using a hydrophilic interaction chromatography based solid-phase extraction and analyzed by RP-LC-ESI-MS. We used our method to show for the first time that AGP N-glycan profile is stable in healthy individuals (14 individuals in 3 time points), which is a requirement for evaluation of its diagnostic potential. Furthermore, we tested our method on a population including individuals with registered hyperglycemia in critical illness (59 cases and 49 controls), which represents a significantly increased risk of developing type 2 diabetes. Individuals at higher risk of diabetes presented increased N-glycan branching on AGP’s second glycosylation site and lower sialylation of N-glycans on AGP’s third and AGP1’s fourth glycosylation site. Although this should be confirmed on a larger prospective cohort, it indicates that site-specific AGP N-glycan profile could help distinguish individuals who are at risk of type 2 diabetes.

scholarly journals Factors affecting the physical stability (aggregation) of peptide therapeutics

2017 ◽  
Vol 7 (6) ◽  
pp. 20170030 ◽  
Author(s):  
Karolina L. Zapadka ◽  
Frederik J. Becher ◽  
A. L. Gomes dos Santos ◽  
Sophie E. Jackson
Keyword(s):  
Current Knowledge ◽  
Physical Stability ◽  
Therapeutic Agents ◽  
Chemical Degradation ◽  
Physical Parameters ◽  
Net Charge ◽  
Factors Affecting ◽  
Surfaces And Interfaces ◽  
Development Pipeline ◽  
Self Association

The number of biological therapeutic agents in the clinic and development pipeline has increased dramatically over the last decade and the number will undoubtedly continue to increase in the coming years. Despite this fact, there are considerable challenges in the development, production and formulation of such biologics particularly with respect to their physical stabilities. There are many cases where self-association to form either amorphous aggregates or highly structured fibrillar species limits their use. Here, we review the numerous factors that influence the physical stability of peptides including both intrinsic and external factors, wherever possible illustrating these with examples that are of therapeutic interest. The effects of sequence, concentration, pH, net charge, excipients, chemical degradation and modification, surfaces and interfaces, and impurities are all discussed. In addition, the effects of physical parameters such as pressure, temperature, agitation and lyophilization are described. We provide an overview of the structures of aggregates formed, as well as our current knowledge of the mechanisms for their formation.

scholarly journals CD63 Promotes Hemocyte-Mediated Phagocytosis in the Clam,Paphia undulata

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Mingjia Yu ◽  
Shanjun Yang ◽  
Hongxia Sun ◽  
Qiang Xia
Keyword(s):  
Profile Analysis ◽  
Surface Membrane ◽  
Bioinformatic Analysis ◽  
Glycosylation Site ◽  
Marine Mollusk ◽  
Reading Frame ◽  
Expression Profile Analysis ◽  
Acid Protein ◽  
Large Extracellular Loop

As one of the surface membrane proteins of tetraspanin family, CD63 plays a crucial role in cellular trafficking and endocytosis, which also is associated with activation of a wide variety of immune cells. Here, the homolog of CD63 was characterized from one marine mollusk,Paphia undulata, which is designated as Pu-CD63. The complete cDNA of Pu-CD63 is 1,738 bp in length with an open reading frame (ORF) of 849 bp, encoding a 282 amino acid protein with four putative hydrophobic transmembrane helixes. Bioinformatic analysis revealed that Pu-CD63 contains one putative YXXØ consensus motif of “110-YVII-113” and one N-glycosylation site “155-NGT-157” within the large extracellular loop (LEL) region, supporting its conserved function in plasma membrane and endosomal/lysosomal trafficking. Moreover, temporal expression profile analysis demonstrates a drastic induction in the expression of CD63 in hemocytes after pathogenic challenge with eitherV. parahaemolyticusorV. alginolyticus. By performing dsRNA-mediate RNAi knockdowns of CD63, a dramatic reduction in hemocytes phagocytic activity to pathogenicVibriois recorded by flow cytometry, revealing the definite role of Pu-CD63 in promoting hemocyte-mediated phagocytosis. Therefore, our work has greatly enhanced our understanding about primitive character of innate immunity in marine mollusk.

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