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1.
MAbs ; 11(1): 94-105, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30570405

RESUMEN

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.


Asunto(s)
Anticuerpos Monoclonales/química , Biofarmacia/normas , Laboratorios/normas , Espectroscopía de Resonancia Magnética/métodos , Humanos , Reproducibilidad de los Resultados
2.
J Eukaryot Microbiol ; 64(2): 164-172, 2017 03.
Artículo en Inglés | MEDLINE | ID: mdl-27455370

RESUMEN

Euplotes is diversified into dozens of widely distributed species that produce structurally homologous families of water-borne protein pheromones governing self-/nonself-recognition phenomena. Structures of pheromones and pheromone coding genes have so far been studied from species lying in different positions of the Euplotes phylogenetic tree. We have now cloned the coding genes and determined the NMR molecular structure of four pheromones isolated from Euplotes petzi, a polar species which is phylogenetically distant from previously studied species and forms the deepest branching clade in the tree. The E. petzi pheromone genes have significantly shorter sequences than in other congeners, lack introns, and encode products of only 32 amino acids. Likewise, the three-dimensional structure of the E. petzi pheromones is markedly simpler than the three-helix up-down-up architecture previously determined in another polar species, Euplotes nobilii, and in a temperate-water species, Euplotes raikovi. Although sharing the same up-down-up architecture, it includes only two short α-helices that find their topological counterparts with the second and third helices of the E. raikovi and E. nobilii pheromones. The overall picture that emerges is that the evolution of Euplotes pheromones involves progressive increases in the gene sequence length and in the complexity of the three-dimensional molecular structure.


Asunto(s)
Euplotes/genética , Euplotes/metabolismo , Sistemas de Lectura Abierta/genética , Feromonas/química , Feromonas/genética , Conformación Proteica , Secuencia de Aminoácidos , Secuencia de Bases , Biodiversidad , Técnicas de Cultivo de Célula , Clima Frío , Frío , ADN Protozoario , Euplotes/clasificación , Evolución Molecular , Genes Protozoarios , Vectores Genéticos , Resonancia Magnética Nuclear Biomolecular/métodos , Feromonas/aislamiento & purificación , Filogenia , Proteínas Protozoarias/genética , Agua de Mar/parasitología , Alineación de Secuencia , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos
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