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Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because 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.
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Anticuerpos Monoclonales/química , Productos Biológicos , Biofarmacia/métodos , Anticuerpos Monoclonales/metabolismo , Glicómica/métodos , Glicopéptidos/metabolismo , Glicosilación , Humanos , Laboratorios , Polisacáridos/metabolismo , Procesamiento Proteico-Postraduccional , Proteómica/métodosRESUMEN
The glycosylation of biologics is an important factor in pharmacological functions such as efficacy, safety, and biological activity and is easily affected by subtle changes in the cellular environment. Hence, comprehensive and in-depth glycomic characterization of biological products should be performed to ensure product quality and process consistency prior to regulatory approval, but it is still highly challenging due to glycan microheterogeneity produced by enzymatic machinery. In this study, we have developed a systematic methodology for the separation and characterization of various glycans of biotherapeutics using the combination of solid-phase extraction (SPE) and high resolution LC/MS. Neutral and multiple-acidic glycans were selectively fractionated by SPE with a porous graphitized carbon (PGC) cartridge according to their molecule size and polarity (acidity, p Ka). Subsequent LC-MS and -MS/MS analyses enabled us to obtain glycan compositions, structures, and quantitative information. Indeed, we have successfully performed glycomic characterization of agalsidase-beta, a representative therapeutic enzyme containing both phosphorylated and sialylated glycans. In addition, a comparative analysis of functional glycans released from different batches of enzymes was performed to verify our method. These results suggest that stepwise PGC-SPE and LC/MS/MS pairwise assays can be used as an efficient tool to detect glycosylation changes of therapeutic glycoproteins including abundant acidic species in biologics or biosimilar development.
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Carbono/química , Polisacáridos/aislamiento & purificación , Extracción en Fase Sólida , Cromatografía Liquida , Humanos , Tamaño de la Partícula , Polisacáridos/química , Porosidad , Propiedades de Superficie , Espectrometría de Masas en TándemRESUMEN
PURPOSE: This qualitative study aimed to explore the lived and true meaning of experiences of middle-aged patients with complex regional pain syndrome. METHODS: The participants were 10 men and women aged 40 to 60 years who received outpatient treatment at a university hospital, could communicate, and agreed to participate in the study. Data were collected through individual interviews using open and semi-structured questions from September 2019 to July 2021 and were analyzed using the content analysis method suggested by Hsieh and Shannon (2005). RESULTS: As a result of this study, 42 summarized semantic units related to life experience, 15 subthemes, and seven themes were derived. The seven themes were "pressed by severe pain," "frustrated because I cannot be part of the community," "distressed because people do not recognize my disease," "sad about conflicts with family," "unmotivated because of desperate life," "appreciating for support," and "putting oneself together and living daily life." CONCLUSION: The vivid experiences of the participants derived in this study are the basic data for developing treatment guidelines. In the future, we propose a study on life and family care experiences according to the developmental characteristics of the life cycle of patients with complex regional pain syndrome and develop and apply programs to support patients and their families.
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Síndromes de Dolor Regional Complejo , Dolor , Persona de Mediana Edad , Masculino , Humanos , Femenino , Investigación Cualitativa , Emociones , Acontecimientos que Cambian la VidaRESUMEN
Lipids are crucial components of cellular function owing to their role in membrane formation, intercellular signaling, energy storage, and homeostasis maintenance. In the brain, lipid dysregulations have been associated with the etiology and progression of neurodegeneration and other neurological pathologies. Hence, brain lipids are emerging as important potential targets for the early diagnosis and prognosis of neurological diseases. This review aims to highlight the significance and usefulness of lipidomics in diagnosing and treating brain diseases. We explored lipid alterations associated with brain diseases, paying attention to organ-specific characteristics and the functions of brain lipids. As the recent advances in brain lipidomics would have been impossible without advances in analytical techniques, we provide up-to-date information on mass spectrometric approaches and integrative analysis with other omic approaches. Last, we present the potential applications of lipidomics combined with artificial intelligence techniques and interdisciplinary collaborative research for treating brain diseases with clinical heterogeneities.
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Encefalopatías , Lipidómica , Inteligencia Artificial , Encéfalo , Encefalopatías/diagnóstico , Encefalopatías/etiología , Humanos , Metabolismo de los Lípidos , Lípidos/químicaRESUMEN
Glycosylation of biologics, an important factor in pharmacological functions such as efficacy, safety, and biological activity, is easily affected by subtle changes in the cellular environment. Therefore, comprehensive and in-depth glycan characterization of therapeutic glycoproteins should be performed to ensure product quality and process consistency, but it is analytically challenging due to glycan microheterogeneity occurring in the glycan biosynthesis pathway. LC-based chromatographic separation combined with mass spectrometry (MS) has been widely used as a prominent tool for the qualitative and quantitative analysis of glycosylation of therapeutic glycoproteins. However, prior to LC/MS analysis, glycans are selectively captured and fractionated by solid-phase extraction (SPE) utilizing physicochemical characteristics for comprehensive characterization of a wide range of glycan heterogeneity on glycoengineered therapeutic proteins. In particular, porous graphitized carbon (PGC) SPE has been employed as a useful technique for the fractionation of native glycans having different sizes and polarities. Here, we describe a systematic method for comprehensive glycan characterization of therapeutic proteins using stepwise PGC SPE and LC/MS.
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Cromatografía Liquida , Glicoproteínas/análisis , Grafito/química , Infliximab/análisis , Isoenzimas/análisis , Espectrometría de Masas , Procesamiento Proteico-Postraduccional , Extracción en Fase Sólida , alfa-Galactosidasa/análisis , Glicoproteínas/uso terapéutico , Glicosilación , Infliximab/uso terapéutico , Isoenzimas/uso terapéutico , Porosidad , Proyectos de Investigación , Flujo de Trabajo , alfa-Galactosidasa/uso terapéuticoRESUMEN
Neural stem cells (NSCs) are multipotent cells capable of self-renewal and differentiation into different nervous system cells. Mouse NSCs (mNSCs) are useful tools for studying neurogenesis and the therapeutic applications of neurodegenerative diseases in mammals. Formyl peptide receptor 2 (FPR2), expressed in the central nervous system and brain, is involved in the migration and differentiation of murine embryonic-derived NSCs. In this study, we explored the effect of FPR2 activation in adult mNSCs using the synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm), an agonist of FPR2. After isolation of NSCs from the subventricular zone of the adult mouse brain, they were cultured in two culture systems-neurospheres or adherent monolayers-to demonstrate the expression of NSC markers and phenotypes. Under different conditions, mNSCs differentiated into neurons and glial cells such as astrocytes, microglia, and oligodendrocytes. Treatment with WKYMVm stimulated the chemotactic migration of mNSCs. Moreover, WKYMVm-treated mNSCs were found to promote proliferation; this result was confirmed by the expansion of mNSCs in Matrigel and the increase in the number of Ki67-positive cells. Incubation of mNSCs with WKYMVm in a supplement-free medium enhanced the survival rate of the mNSCs. Together, these results suggest that WKYMVm-induced activation of FPR2 stimulates cellular responses in adult NSCs.
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Proteomics has become an important field in molecular sciences, as it provides valuable information on the identity, expression levels, and modification of proteins. For example, cancer proteomics unraveled key information in mechanistic studies on tumor growth and metastasis, which has contributed to the identification of clinically applicable biomarkers as well as therapeutic targets. Several cancer proteome databases have been established and are being shared worldwide. Importantly, the integration of proteomics studies with other omics is providing extensive data related to molecular mechanisms and target modulators. These data may be analyzed and processed through bioinformatic pipelines to obtain useful information. The purpose of this review is to provide an overview of cancer proteomics and recent advances in proteomic techniques. In particular, we aim to offer insights into current proteomics studies of brain cancer, in which proteomic applications are in a relatively early stage. This review covers applications of proteomics from the discovery of biomarkers to the characterization of molecular mechanisms through advances in technology. Moreover, it addresses global trends in proteomics approaches for translational research. As a core method in translational research, the continued development of this field is expected to provide valuable information at a scale beyond that previously seen.
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Limitation of current anti-Vascular Endothelial Growth Factor (VEGF) cancer therapy is transitory responses, inevitable relapses and its insufficient tumor-targeting. Thus, multifaceted approaches, including the development of bispecific antibodies and combination strategies targeting different pathways have been proposed as an alternative. Here, we developed a novel multi-paratopic VEGF decoy receptor, Cetuximab-VEGF-Grab and Trastuzumab-VEGF-Grab, by genetically fusing VEGF decoy receptor (VEGF-Grab) to a single chain Fv of anti-Epidermal Growth Factor Receptor (EGFR) antibody (Cetuximab and Trastuzumab). These multi-paratopic VEGF decoy receptor, which recognize VEGF and EGFR family (EGFR or HER2), effectively suppressed both VEGF and EGFR pathways in vitro, to levels similar to those of the parental VEGF-Grab and anti-EGFR antibodies. In addition, the concurrent binding of multi-paratopic VEGF decoy receptor to VEGF and EGFR family enabled their specific localization to EGFR + tumor in vitro and in vivo. Furthermore, Cetuximab-VEGF-Grab and Trastuzumab-VEGF-Grab exhibited the enhanced anti-tumor activities compared to VEGF-Grab in EGFR + tumor xenograft mouse model via anti-EGFR and the targeted anti-angiogenic activities. These results indicate that multi-paratopic VEGF decoy receptor can be a promising agent, combining tumor-targeted anti-angiogenic therapy with efficient blockade of proliferative signals mediated by EGFR family.
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Inhibidores de la Angiogénesis/uso terapéutico , Antineoplásicos/uso terapéutico , Receptores ErbB/antagonistas & inhibidores , Neovascularización Patológica/tratamiento farmacológico , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Inhibidores de la Angiogénesis/farmacología , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones Desnudos , Neovascularización Fisiológica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
OBJECTIVE: To identify whether somatic mutations in SLC35A2 alter N-glycan structures in human brain tissues and cause nonlesional focal epilepsy (NLFE) or mild malformation of cortical development (mMCD). METHODS: Deep whole exome and targeted sequencing analyses were conducted for matched brain and blood tissues from patients with intractable NLFE and patients with mMCD who are negative for mutations in mTOR pathway genes. Furthermore, tissue glyco-capture and nanoLC/mass spectrometry analysis were performed to examine N-glycosylation in affected brain tissue. RESULTS: Six of the 31 (19.3%) study patients exhibited brain-only mutations in SLC35A2 (mostly nonsense and splicing site mutations) encoding a uridine diphosphate (UDP)-galactose transporter. Glycome analysis revealed the presence of an aberrant N-glycan series, including high degrees of N-acetylglucosamine, in brain tissues with SLC35A2 mutations. CONCLUSION: Our study suggests that brain somatic mutations in SLC35A2 cause intractable focal epilepsy with NLFE or mMCD via aberrant N-glycosylation in the affected brain.
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AIM: Recombinant coagulation factor IX (rFIX) has extraordinarily multiple post-translational modifications including N-glycosylation and O-glycosylation which have a drastic effect on biological functions and in vivo recovery. Unlike N-glycosylation extensively characterized, there are a few studies on O-glycosylation due to its intrinsic complexity. In-depth O-glycosylation analysis is necessary to better understand and assess pharmacological activity of rFIX. RESULTS: We determined unusual O-glycosylations including O-fucosylation and O-glucosylation which were located at Serine 53 and 61, respectively in EGF domain. Other O-glycosylations bearing core 1 glycan moiety were found on activation peptide. CONCLUSION: This is the first comprehensive study to characterize O-glycosylation of rFIX using MS-based glycomic and glycoproteomic approaches. Site-specific profiling will be a powerful platform to determine bioequivalence of biosimilars.
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Factor IX/metabolismo , Glicómica , Proteínas Recombinantes/metabolismo , Cromatografía Liquida , Glicosilación , Humanos , Espectrometría de Masas en TándemRESUMEN
AIM: Glycosylation of recombinant human erythropoietins (rhEPOs) is significantly associated with drug's quality and potency. Thus, comprehensive characterization of glycosylation is vital to assess the biotherapeutic quality and establish the equivalency of biosimilar rhEPOs. However, current glycan analysis mainly focuses on the N-glycans due to the absence of analytical tools to liberate O-glycans with high sensitivity. We developed selective and sensitive method to profile native O-glycans on rhEPOs. RESULTS: O-glycosylation on rhEPO including O-acetylation on a sialic acid was comprehensively characterized. Details such as O-glycan structure and O-acetyl-modification site were obtained from tandem MS. CONCLUSION: This method may be applied to QC and batch analysis of not only rhEPOs but also other biotherapeutics bearing multiple O-glycosylations.
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Eritropoyetina/metabolismo , Límite de Detección , Oxígeno/metabolismo , Eritropoyetina/química , Eritropoyetina/uso terapéutico , Glicómica , Glicosilación , Humanos , Ácido N-Acetilneuramínico/metabolismoRESUMEN
The presence of endotoxin in water environments may pose a serious public health hazard. We investigated the effectiveness of advanced oxidative processes (AOP: O3/H2O2 and UV/H2O2) in the oxidative degradation of endotoxin. In addition, we measured the release of endotoxin from Escherichia coli following typical disinfection methods, such as chlorine, ozone alone and UV, and compared it with the use of AOPs. Finally, we tested the AOP-treated samples in their ability to induce tumor necrosis factor alpha (TNF-α) in mouse peritoneal macrophages. The production of hydroxyl radical in AOPs showed superior ability to degrade endotoxin in buffered solution, as well as water samples from Korean water treatment facilities, with the ozone/H2O2 being more efficient compared to UV/H2O2. In addition, the AOPs proved effective not only in eliminating E. coli in the samples, but also in endotoxin degradation, while the standard disinfection methods lead to the release of endotoxin following the bacteria destruction. Furthermore, in the experiments with macrophages, the AOPs-deactivated endotoxin lead to the smallest induction of TNF-α, which shows the loss of inflammation activity, compared to ozone treatment alone. In conclusion, these results suggest that AOPs offer an effective and mild method for endotoxin degradation in the water systems.