NIST interlaboratory study on glycosylation analysis of monoclonal antibodies : comparison of results from diverse analytical methods

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals since it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy‑six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation  analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type.. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods

Revising the embryonic origin of thyroid C cells in mice and humans.

Current understanding infers a neural crest origin of thyroid C cells, the major source of calcitonin in mammals and ancestors to neuroendocrine thyroid tumors. The concept is primarily based on investigations in quail–chick chimeras involving fate mapping of neural crest cells to the ultimobranchial glands that regulate Ca2+ homeostasis in birds, reptiles, amphibians and fishes, but whether mammalian C cell development involves a homologous ontogenetic trajectory has not been experimentally verified. With lineage tracing, we now provide direct evidence that Sox17+ anterior endoderm is the only source of differentiated C cells and their progenitors in mice. Like many gut endoderm derivatives, embryonic C cells were found to coexpress pioneer factors forkhead box (Fox) a1 and Foxa2 before neuroendocrine differentiation takes place. In the ultimobranchial body epithelium emerging from pharyngeal pouch endoderm in early organogenesis, differential Foxa1/Foxa2 expression distinguished two spatially separated pools of C cell precursors with different growth properties. A similar expression pattern was recapitulated in medullary thyroid carcinoma cells in vivo, consistent with a growth-promoting role of Foxa1. In contrast to embryonic precursor cells, C cell-derived tumor cells invading the stromal compartment downregulated Foxa2, foregoing epithelial-to-mesenchymal transition designated by loss of E-cadherin;bothFoxa2 andE-cadherinwere re-expressed atmetastatic sites. These findings revise mammalian C cell ontogeny, expand the neuroendocrine repertoire of endoderm and redefine the boundaries of neural crest diversification.The data further underpin distinct functions of Foxa1 and Foxa2 in both embryonic and tumor development

Revising the embryonic origin of thyroid C cells in mice and humans

Current understanding infers a neural crest origin of thyroid C cells, the major source of calcitonin in mammals and ancestors to neuroendocrine thyroid tumors. The concept is primarily based on investigations in quail–chick chimeras involving fate mapping of neural crest cells to the ultimobranchial glands that regulate Ca2+ homeostasis in birds, reptiles, amphibians and fishes, but whether mammalian C cell development involves a homologous ontogenetic trajectory has not been experimentally verified. With lineage tracing, we now provide direct evidence that Sox17+ anterior endoderm is the only source of differentiated C cells and their progenitors in mice. Like many gut endoderm derivatives, embryonic C cells were found to coexpress pioneer factors forkhead box (Fox) a1 and Foxa2 before neuroendocrine differentiation takes place. In the ultimobranchial body epithelium emerging from pharyngeal pouch endoderm in early organogenesis, differential Foxa1/Foxa2 expression distinguished two spatially separated pools of C cell precursors with different growth properties. A similar expression pattern was recapitulated in medullary thyroid carcinoma cells in vivo, consistent with a growth-promoting role of Foxa1. In contrast to embryonic precursor cells, C cell-derived tumor cells invading the stromal compartment downregulated Foxa2, foregoing epithelial-to-mesenchymal transition designated by loss of E-cadherin;bothFoxa2 andE-cadherinwere re-expressed atmetastatic sites. These findings revise mammalian C cell ontogeny, expand the neuroendocrine repertoire of endoderm and redefine the boundaries of neural crest diversification.The data further underpin distinct functions of Foxa1 and Foxa2 in both embryonic and tumor development