112 research outputs found
high resolution glycoform profiling of intact therapeutic proteins by hydrophilic interaction chromatography mass spectrometry
Abstract Glycosylation is considered a critical quality attribute of therapeutic proteins. Protein heterogeneity introduced by glycosylation includes differences in the nature, number and position of the glycans. Whereas analysis of released glycans and glycopeptides provides information about the composition and/or position of the glycan, intact glycoprotein analysis allows assignment of individual proteoforms and co-occurring modifications. Yet, resolving protein glycoforms at the intact level is challenging. We have explored the capacity of hydrophilic liquid chromatography-mass spectrometry (HILIC-MS) for assessing glycosylation patterns of intact pharmaceutical proteins by analyzing the complex glycoproteins interferon-beta-1a (rhIFN-βâŻââŻ1a) and recombinant human erythropoietin (rhEPO). Efficient glycoform separation was achieved using a superficially-porous amide HILIC stationary phase and trifluoroacetic acid (TFA) as eluent additive. In-source collision-induced dissociation proved to be very useful to minimize protein-signal suppression effects by TFA. Direct injection of therapeutic proteins in aqueous formulation was possible without causing extra band dispersion, provided that the sample injection volume was not larger than 2âŻÎźL. HILIC-MS of rhIFN-βâŻââŻ1a and rhEPO allowed the assignment of, respectively, 15 and 51 glycoform compositions, next to a variety of posttranslational modifications, such as succinimide, oxidation and N-terminal methionine-loss products. MS-based assignments showed that neutral glycan units significantly contributed to glycoform separation, whereas terminal sialic acids only had a marginal effect on HILIC retention. Comparisons of HILIC-MS with the selectivity provided by capillary electrophoresis-MS for the same glycoproteins, revealed a remarkable complementarity of the techniques. Finally it was demonstrated that by replacing TFA for difluoroacetic acid, peak resolution somewhat decreased, but rhEPO glycoforms with relative abundances below 1% could be detected by HILIC-MS, increasing the overall rhEPO glycoform coverage to 72
Glycovaccine Design: Optimization of Model and Antitubercular Carrier Glycosylation via Disuccinimidyl Homobifunctional Linker
Conjugation via disuccinimidyl homobifunctional linkers is reported in the literature as a convenient approach for the synthesis of glycoconjugate vaccines. However, the high tendency for hydrolysis of disuccinimidyl linkers hampers their extensive purification, which unavoidably results in side-reactions and non-pure glycoconjugates. In this paper, conjugation of 3-aminopropyl saccharides via disuccinimidyl glutarate (DSG) was exploited for the synthesis of glycoconjugates. A model protein, ribonuclease A (RNase A), was first considered to set up the conjugation strategy with mono- to tri- mannose saccharides. Through a detailed characterization of synthetized glycoconjugates, purification protocols and conjugation conditions have been revised and optimized with a dual aim: ensure high sugar-loading and avoid the presence of side reaction products. An alternative purification approach based on hydrophilic interaction liquid chromatography (HILIC) allowed the formation of glutaric acid conjugates to be avoided, and a design of experiment (DoE) approach led to optimal glycan loading. Once its suitability was proven, the developed conjugation strategy was applied to the chemical glycosylation of two recombinant antigens, native Ag85B and its variant Ag85B-dm, that are candidate carriers for the development of a novel antitubercular vaccine. Pure glycoconjugates (âĽ99.5%) were obtained. Altogether, the results suggest that, with an adequate protocol, conjugation via disuccinimidyl linkers can be a valuable approach to produce high sugar-loaded and well-defined glycovaccines
Proteomic analysis of small acid soluble proteins in the spore core of Bacillus subtilis ÎprpE and 168 strains with predictions of peptides liquid chromatography retention times as an additional tool in protein identification
Immobilizzazione di enzimi e mutagenesi sito-specifica
Lâimmobilizzazione di enzimi su supporto solido consente di ottenere biocatalizzatori insolubili facilmente recuperabili dallâambiente di reazione, riutilizzabili e caratterizzati da maggiore stabilitĂ .
Per evitare approcci dispendiosi del tipo âtrial and
errorâ nella scelta del supporto e della tecnica
di immobilizzazione, è fondamentale integrare
le informazioni strutturali sulla proteina con le
proprietĂ chimico-fisiche dei carrier utilizzati
per lâimmobilizzazione. Lâimportanza di questa
strategia è stata confermata da tecniche di
mutagenesi sito-specifica e di analisi LC-MS/MS
utilizzate per determinare lâorientamento dellâenzima
sul supporto, descrittivo del grado di accessibilitĂ
del sito attivo da parte dei substrati, e quindi
dellâattivitĂ dellâenzima immobilizzato
Penicillin G acylase as chiral selector in LC and CE: exploring the origins of enantioselectivity
Protein-Labs on Separative Analytical Scale in Medicinal Chemistry: from the Proof of Concept to Applications
Coupling of a macromolecule such as an enzyme or a receptor to a chromatographic support has revealed a powerful analytical tool to face different aspects related to drug discovery. Chromatographic systems with immobilized macromolecules have been developed to be used for different applications in pharmaceutical analysis including enzyme inhibitor screening, biopharmaceutics structural analysis, chiral separations and binding studies. These areas of research are very challenging both for the technological and for the applicative aspects. This review offers an overview of general strategies and references that can be considered in order to develop innovative and reliable âprotein-labsâ for specific separative analytical applications of pharmaceutical interest
Strategy and realization of chromatographic tools for ligand discovery by frontal affinity chromatography-mass spectrometry (comunicazione orale)
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