scholarly journals Structural and In Vitro Functional Comparability Analysis of Altebrel™, a Proposed Etanercept Biosimilar: Focus on Primary Sequence and Glycosylation

2019 ◽  
Vol 12 (1) ◽  
pp. 14 ◽  
Author(s):  
Ramin Fazel ◽  
Yudong Guan ◽  
Behrouz Vaziri ◽  
Christoph Krisp ◽  
Laura Heikaus ◽  
...  
Keyword(s):  
Primary Structure ◽  
Peptide Mapping ◽  
Spectrometric Analysis ◽  
Methionine Oxidation ◽  
Primary Sequence ◽  
Surface Plasmon Resonance Analysis ◽  
Resonance Analysis ◽  
Glycosylation Sites ◽  
Primary Sequence Analysis

The demand for reliable comparability studies of biosimilars grows with their increased market share. These studies focus on physicochemical, structural, functional and clinical properties to ensure that a biosimilar has no significant differences to the originator product and can be released into the market without extensive clinical trials. In the current study, Enbrel® (etanercept, the originator) and Altebrel™ (the proposed biosimilar) underwent direct comparison. “Bottom-up” mass spectrometric analysis was used for primary sequence analysis, evaluation of N/O-glycosylation sites and quantification of methionine oxidation. N/O-glycans were analyzed after permethylation derivatization and the effect of N-glycans on in-vitro functionality of etanercept was assayed. Three enzyme peptide mapping resulted in complete identification of the primary structure. It was confirmed that total ion chromatograms are valuable datasets for the analysis of the primary structure of biodrugs. New N/O-glycan structures were identified and all the N-glycans were quantified. Finally, investigation of the functional properties of N-deglycosylated and non-modified etanercept samples using surface plasmon resonance analysis and in-vitro bioassay showed that N-glycosylation has no significant effect on its in-vitro functionality. Analysis of etanercept and its biosimilar, revealed a high similarity in terms of glycosylation, primary structure and in-vitro functionality.

scholarly journals Differential Effects of Paromomycin on Ribosomes ofLeishmania mexicanaand Mammalian Cells

2010 ◽  
Vol 55 (1) ◽  
pp. 86-93 ◽  
Author(s):  
Marisa M. Fernández ◽  
Emilio L. Malchiodi ◽  
Israel D. Algranati
Keyword(s):  
Protein Synthesis ◽  
Mammalian Cell ◽  
Mammalian Cells ◽  
Aminoglycoside Antibiotic ◽  
Surface Plasmon Resonance Analysis ◽  
Differential Effects ◽  
Resonance Analysis ◽  
Neomycin B

ABSTRACTParomomycin, an aminoglycoside antibiotic having low mammalian cell toxicity, is one of the drugs currently used in the chemotherapy of cutaneous and visceral leishmaniasis. In order to understand the mode of action of this antibiotic at the molecular level, we have investigated the effects of paromomycin on protein synthesis inLeishmaniaand its mammalian hosts. We were able to demonstrate thatin vivoprotein synthesis in the promastigote stage of the parasite and its proliferation rate are markedly inhibited by paromomycin while being only slightly affected by other aminoglycoside antibiotics, such as streptomycin and neomycin B. Furthermore, bothin vitropolypeptide synthesis induced by poly(U) as mRNA and accuracy of translation are significantly decreased by paromomycin in cell-free systems containing ribosomal particles ofLeishmaniapromastigotes. Conversely, when ribosomes from mammalian cells are used instead of the protozoan particles, polyphenylalanine synthesis is only barely reduced by the antibiotic and the translation misreading remains almost unaltered. Surface plasmon resonance analysis of the interaction between paromomycin and protozoan or mammalian cell ribosomal RNAs shows a strong binding of antibiotic to the parasite ribosomal decoding site and practically no interaction with the mammalian cell counterpart. Our results indicating differential effects of paromomycin on the translation processes of theLeishmaniaparasite and its mammalian hosts can explain the therapeutic efficiency of this antibiotic as an antileishmaniasis agent.

scholarly journals Antibodies produced in vitro in the detection of periodontal bacteria by using surface plasmon resonance analysis

2015 ◽  
Vol 1 (1) ◽  
pp. 32-44 ◽  
Author(s):  
Sravya Sowdamini Nakka ◽  
Johanna Lönn ◽  
Carin Starkhammar Johansson ◽  
Torbjörn Bengtsson ◽  
Fariba Nayeri
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Surface Plasmon Resonance Analysis ◽  
Resonance Analysis ◽  
Periodontal Bacteria

scholarly journals Novel Approach to Analyzing RNA Aptamer-Protein Interactions: Toward Further Applications of Aptamers

2006 ◽  
Vol 11 (6) ◽  
pp. 599-605 ◽  
Author(s):  
Joonsung Hwang ◽  
Satoshi Nishikawa
Keyword(s):  
Protein Interactions ◽  
In Vitro Selection ◽  
Selection Procedure ◽  
Rna Aptamers ◽  
Sensor Chip ◽  
Rna Aptamer ◽  
Surface Plasmon Resonance Analysis ◽  
Resonance Analysis ◽  
Novel Approach

Surface plasmon-resonance analysis using a Biacore biosensor is a powerful tool for the detailed study of biomolecular interactions. The authors examined the methods of immobilizing proteins on the surface of NTA, SA, and CM5 sensor chips to study RNA aptamer-protein interactions. RNA aptamers and their deletion variants were loaded onto a protein-immobilized sensor chip, and their binding affinities were analyzed. Immobilizing the protein on a CM5 sensor chip via an anti-His-tag antibody was the only strategy that clearly detected the kinetic parameters of the interactions. ΔNEO-III-14U, one of the deletion variants of the NS3 aptamer, had the highest binding affinity for the ΔNS3 protein in this study (KD = 4 × 10-8). Moreover, the 29-amino-acid spacer fragment was essential for protein immobilization using this strategy. This novel method will be useful in comparing the affinity of various RNA aptamers and selecting the most suitable candidates for a given target, as well as facilitating the in vitro selection procedure itself.

scholarly journals Holospiniferoside: A New Antitumor Cerebroside from The Red Sea Cucumber Holothuria spinifera: In Vitro and In Silico Studies

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1555
Author(s):  
Enas E. Eltamany ◽  
Usama Ramadan Abdelmohsen ◽  
Dina M. Hal ◽  
Amany K. Ibrahim ◽  
Hashim A. Hassanean ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Red Sea ◽  
Active Site ◽  
Sea Cucumber ◽  
Gas Chromatography Mass Spectrometry ◽  
Spectrometric Analysis ◽  
Chemical Structures ◽  
In Silico Studies ◽  
Red Sea Cucumber

Chemical investigation of the methanolic extract of the Red Sea cucumber Holothuria spinifera led to the isolation of a new cerebroside, holospiniferoside (1), together with thymidine (2), methyl-α-d-glucopyranoside (3), a new triacylglycerol (4), and cholesterol (5). Their chemical structures were established by NMR and mass spectrometric analysis, including gas chromatography–mass spectrometry (GC–MS) and high-resolution mass spectrometry (HRMS). All the isolated compounds are reported in this species for the first time. Moreover, compound 1 exhibited promising in vitro antiproliferative effect on the human breast cancer cell line (MCF-7) with IC50 of 20.6 µM compared to the IC50 of 15.3 µM for the drug cisplatin. To predict the possible mechanism underlying the cytotoxicity of compound 1, a docking study was performed to elucidate its binding interactions with the active site of the protein Mdm2–p53. Compound 1 displayed an apoptotic activity via strong interaction with the active site of the target protein. This study highlights the importance of marine natural products in the design of new anticancer agents.

scholarly journals Studies on the autophosphorylation of the insulin receptor from human placenta. Analysis of the sites phosphorylated by two-dimensional peptide mapping

1988 ◽  
Vol 252 (2) ◽  
pp. 607-615 ◽  
Author(s):  
J M Tavaré ◽  
R M Denton
Keyword(s):  
Thin Layer ◽  
Insulin Receptor ◽  
Peptide Mapping ◽  
Beta Subunit ◽  
Two Dimensional ◽  
Intact Cells ◽  
Tyrosine Residues ◽  
Domain 3 ◽  
Receptor Beta

1. A partially purified preparation of human placental insulin receptors was incubated with [gamma-32P]ATP in the presence or absence of insulin. The 32P-labelled insulin-receptor beta-subunits were then isolated, cleaved with trypsin followed by protease V8 and the [32P]phosphopeptides generated were analysed by thin layer electrophoresis and chromatography. This approach revealed that insulin stimulates autophosphorylation of the insulin-receptor beta-subunit in vitro on at least seven tyrosine residues distributed among three distinct domains. 2. One domain (domain 2), containing tyrosine residues 1146, 1150 and 1151 was the most rapidly phosphorylated and could be recovered as mono-, di- and triphosphorylated peptides cleaved by trypsin at Arg-1143 and either Lys-1153 or Lys-1156. Multiple phosphorylation of this domain appears to partially inhibit the cleavage at Lys-1153 by trypsin. 3. In a second domain (domain 3) containing two phosphorylated tyrosine residues at positions 1316 and 1322 the tyrosines were phosphorylated more slowly than those in domain 2. This domain is close to the C-terminus of the beta-subunit polypeptide chain. 4. At least two further tyrosine residues appeared to be phosphorylated after those in domains 2 and 3. These residues probably residue within a domain lying in close proximity to the inner face of the plasma membrane containing tyrosines 953, 960 and 972, but conclusive evidence is still required. 5. The two-dimensional thin-layer analysis employed in this study to investigate insulin-receptor phosphorylation has several advantages over previous methods based on reverse-phase chromatography. It allows greater resolution of 32P-labelled tryptic peptides and, when coupled to radioautography, is considerably more sensitive. The approach can be readily adapted to study phosphorylation of the insulin receptor within intact cells.

scholarly journals Discovery and Characterization of a Novel CD4-Binding Adnectin with Potent Anti-HIV Activity

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
David Wensel ◽  
Yongnian Sun ◽  
Zhufang Li ◽  
Sharon Zhang ◽  
Caryn Picarillo ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Viral Entry ◽  
High Affinity ◽  
Glycosylation Sites ◽  
Spectrum Activity ◽  
Anti Hiv ◽  
Hiv 1 ◽  
Broad Spectrum Activity

ABSTRACT A novel fibronectin-based protein (Adnectin) HIV-1 inhibitor was generated using in vitro selection. This inhibitor binds to human CD4 with a high affinity (3.9 nM) and inhibits viral entry at a step after CD4 engagement and preceding membrane fusion. The progenitor sequence of this novel inhibitor was selected from a library of trillions of Adnectin variants using mRNA display and then further optimized for improved antiviral and physical properties. The final optimized inhibitor exhibited full potency against a panel of 124 envelope (gp160) proteins spanning 11 subtypes, indicating broad-spectrum activity. Resistance profiling studies showed that this inhibitor required 30 passages (151 days) in culture to acquire sufficient resistance to result in viral titer breakthrough. Resistance mapped to the loss of multiple potential N-linked glycosylation sites in gp120, suggesting that inhibition is due to steric hindrance of CD4-binding-induced conformational changes.

scholarly journals Surface plasmon resonance analysis at a supported lipid monolayer

1998 ◽  
Vol 1373 (1) ◽  
pp. 101-111 ◽  
Author(s):  
Matthew A. Cooper ◽  
Andrew C. Try ◽  
Joe Carroll ◽  
David J. Ellar ◽  
Dudley H. Williams
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Lipid Monolayer ◽  
Surface Plasmon Resonance Analysis ◽  
Resonance Analysis

scholarly journals Functional in vitro assessment of modified antibodies: Impact of label on protein properties

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257342
Author(s):  
Martin R. Edelmann ◽  
Simon Hauri
Keyword(s):  
Animal Experiments ◽  
Size Exclusion ◽  
Life Extension ◽  
Spectrometric Analysis ◽  
Pharmacokinetic Studies ◽  
Therapeutic Antibodies ◽  
Surface Binding ◽  
Analytical Strategy ◽  
Protein Properties

Labelling of therapeutic antibodies with radionuclides or fluorophores is routinely used to study their pharmacokinetic properties. A critical assumption in utilizing labelled therapeutic antibodies is that the label has no unfavourable effects on antibody charge, hydrophobicity, or receptor affinity. Ideally, the labelled protein should not have any significant deviations from the physiological properties of the original molecule. This article describes an established quality in vitro assessment workflow for labelled antibodies that ensures better prediction of changes in antibody pharmacokinetic (PK) properties after modifications. This analysis package considers degradation and aggregation analysis by size-exclusion chromatography, changes in neonatal-Fc-receptor (FcRn) affinity, and heparin interaction. FcRn binding is important for antibody recycling and half-life extension, whereas heparin affinity provides estimates on the rate of endocytosis through unspecific cell surface binding. Additionally, mass spectrometric analysis to determine the degree of labelling (DoL) completes the package and the combined analysis data allow to predict the label contribution to the PK properties of the modified antibody. This analytical strategy for labelling 11 IgGs has been investigated using 2 different IgG1 constructs and applying 7 different types of labels. Each labelling resulted in a change in the physicochemical properties of the protein. Not only can the DoL of modified IgGs lead to a change in protein properties, but the type of label also can. Furthermore, it was demonstrated that the labelling process can also influence the behaviour of labelled mAbs. An identical label on different constructs of IgG1 can cause different affinities for FcRn and heparin. Considering the assessment data, only 6 of the 11 modified antibodies from this study can be recommended for subsequent experiments. In conclusion, a suitability assessment of labelled antibodies prior to any pharmacokinetic studies is essential to reduce cost, allocate resources and reduce the number of animal experiments during pre-clinical drug development.

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