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https://www.readbyqxmd.com/read/28346883/identification-and-characterization-of-a-thermally-cleaved-fragment-of-monoclonal-antibody-a-detected-by-sodium-dodecyl-sulfate-capillary-gel-electrophoresis
#1
Kei Kubota, Naoki Kobayashi, Masayuki Yabuta, Motomu Ohara, Toyohiro Naito, Takuya Kubo, Koji Otsuka
This report describes a novel, comprehensive approach to identifying a fragment peak of monoclonal antibody-A (mAb-A), detected by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-cGE). The fragment migrated close to the internal standard (10kDa marker) of SDS-cGE and increased about 0.5% under a 25°C condition for 6 months. Generally, identification of fragments observed in SDS-cGE is challenging to carry out due to the difficulty of collecting analytical amounts of fractionations from the capillary...
March 18, 2017: Journal of Pharmaceutical and Biomedical Analysis
https://www.readbyqxmd.com/read/28341808/protein-coalation-a-redox-regulated-protein-modification-by-coenzyme-ain-mammalian-cells
#2
Yugo Tsuchiya, Sew Peak-Chew, Clare Newell, Sheritta Miller-Aidoo, Sriyash Mangal, Alexander Zhyvoloup, Jovana Baković, Oksana Malanchuk, Gonçalo Pereira, Vassilios Kotiadis, Gyorgy Szabadkai, Michael Duchen, Mark Campbell, Sergio Rodriguez Cuenca, Antonio Vidal-Puig, Andrew James, Michael P Murphy, Valeriy Filonenko, Mark Skehel, Ivan Gout
Coenzyme A (CoA) is an obligatory cofactor in all branches of life. CoA and its derivatives are involved in major metabolic pathways, allosteric interactions and the regulation of gene expression. Abnormal biosynthesis and homeostasis of CoA and its derivatives have been associated with various human pathologies, including cancer, diabetes and neurodegeneration. Using an anti-CoA monoclonal antibody and mass spectrometry we identified a wide range of cellular proteins which are modified by covalent attachment of CoA to cysteine thiols (CoAlation)...
March 24, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28327200/a-first-in-human-phase-1-study-of-a-hepcidin-monoclonal-antibody-ly2787106-in-cancer-associated-anemia
#3
Saroj Vadhan-Raj, Rafat Abonour, Jonathan W Goldman, David A Smith, Christopher A Slapak, Robert L Ilaria, Ramon V Tiu, Xuejing Wang, Sophie Callies, Joanne Cox, Jay L Tuttle, Yiu-Keung Lau, Eric J Roeland
BACKGROUND: Hepcidin plays a central role in iron homeostasis and erythropoiesis. Neutralizing hepcidin with a monoclonal antibody (mAb) may prevent ferroportin internalization, restore iron efflux from cells, and allow transferrin-mediated iron transport to the bone marrow. This multicenter, phase 1 study evaluated the safety, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of a fully humanized mAb (LY2787106) with high affinity for hepcidin in cancer patients with anemia...
March 21, 2017: Journal of Hematology & Oncology
https://www.readbyqxmd.com/read/28274724/characterization-of-succinimide-stability-during-trypsin-digestion-for-lc-ms-analysis
#4
Christine Nowak, Gomathinayagam Ponniah, Alyssa Neill, Hongcheng Liu
LC-MS peptide mapping is the most commonly used method to analyze protein modifications. The proteins are generally digested using trypsin at a slightly basic pH at 37 °C from several hours to overnight. Assay-induced artifacts can be generated during this procedure, potentially causing false-positive or false-negative results for a given modification. Unfortunately, for the analysis of succinimide, both false-negative and false-positive results can be generated within the same procedure. This study evaluates the stability of succinimide during the peptide mapping procedure and has demonstrated that up to 13% of pre-existing succinimide was lost during a 4 h trypsin digestion at pH 5...
March 6, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28259457/optimization-of-ion-exchange-sigmoidal-gradients-using-hybrid-models-implementation-of-quality-by-design-in-analytical-method-development
#5
Varsha S Joshi, Vijesh Kumar, Anurag S Rathore
Thorough product understanding is one of the basic tenets for successful implementation of Quality by Design (QbD). Complexity encountered in analytical characterization of biotech therapeutics such as monoclonal antibodies (mAbs) requires novel, simpler, and generic approaches towards product characterization. This paper presents a methodology for implementation of QbD for analytical method development. Optimization of an analytical cation exchange high performance liquid chromatography (CEX-HPLC) method utilizing a sigmoidal gradient has been performed using a hybrid mechanistic model that is based on Design of experiment (DOE) based studies...
March 31, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28242439/kv3-1-kv3-2-channel-positive-modulators-enable-faster-activating-kinetics-and-increase-firing-frequency-in-fast-spiking-gabaergic-interneurons
#6
Kim Boddum, Charlotte Hougaard, Julie Xiao-Ying Lin, Nadia Lybøl von Schoubye, Henrik Sindal Jensen, Morten Grunnet, Thomas Jespersen
Due to their fast kinetic properties, Kv3.1 voltage gated potassium channels are important in setting and controlling firing frequency in neurons and pivotal in generating high frequency firing of interneurons. Pharmacological activation of Kv3.1 channels may possess therapeutic potential for treatment of epilepsy, hearing disorders, schizophrenia and cognitive impairments. Here we thoroughly investigate the selectivity and positive modulation of the two small molecules, EX15 and RE01, on Kv3 channels. Selectivity studies, conducted in Xenopus laevis oocytes confirmed a positive modulatory effect of the two compounds on Kv3...
February 24, 2017: Neuropharmacology
https://www.readbyqxmd.com/read/28224631/development-and-application-of-monoclonal-antibodies-against-igm-of-black-rockfish-sebastes-schlegeli
#7
W Zhang, X Q Tang, X Z Sheng, J Xing, W B Zhan
Monoclonal antibodies (mAbs) against black rockfish Sebastes schlegeli serum immunoglobulin M (IgM) were developed, which showed a specific reaction with the heavy chain of S. schlegeli IgM in Western blotting and with surface IgM positive (sIgM(+) ) lymphocytes in indirect immunofluorescence. mAb 2A6 was employed to investigate the antibody and sIgM(+) lymphocyte responses of S. schlegeli injected with inactivated Edwardsiella tarda, by ELISA and flow cytometry. Compared with controls, the level of specific antibodies and the percentage of sIgM(+) lymphocytes both increased in the immunized fish and simultaneously reached their peaks at day 35 after immunization...
February 21, 2017: Journal of Fish Biology
https://www.readbyqxmd.com/read/28223726/dendritic-cells-engineered-to-secrete-anti-dcr3-antibody-augment-cytotoxic-t-lymphocyte-response-against-pancreatic-cancer-in-vitro
#8
Jiang Chen, Xiao-Zhong Guo, Hong-Yu Li, Jia-Jun Zhao, Wen-Da Xu
AIM: To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS: DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology...
February 7, 2017: World Journal of Gastroenterology: WJG
https://www.readbyqxmd.com/read/28209962/characterization-of-celiac-disease-related-oat-proteins-bases-for-the-development-of-high-quality-oat-varieties-suitable-for-celiac-patients
#9
María J Giménez, Ana Real, M Dolores García-Molina, Carolina Sousa, Francisco Barro
Some studies have suggested that the immunogenicity of oats depends on the cultivar. RP-HPLC has been proposed as a useful technique to select varieties of oats with reduced immunogenicity. The aim of this study was to identify both the avenin protein patterns associated with low gluten content and the available variability for the development of new non-toxic oat cultivars. The peaks of alcohol-soluble avenins of a collection of landraces and cultivars of oats have been characterized based on the RP-HPLC elution times...
February 17, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28178596/automated-2d-hplc-method-for-characterization-of-protein-aggregation-with-in-line-fraction-collection-device
#10
Abasha Williams, Erik K Read, Cyrus D Agarabi, Scott Lute, Kurt A Brorson
Monoclonal antibodies are mainly produced by mammalian cell culture, which due to its complexity, results in a wide range of product variants/isoforms. With the growing implementation of Quality by Design (QbD) and Process Analytical Technology (PAT) in drug manufacturing, monitoring and controlling quality attributes within a predefined range during manufacturing may provide added consistency to product quality. To implement these concepts, more robust analytical tools could reduce the time needed for monitoring quality attributes during upstream processing...
January 25, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28153228/a-novel-microchip-electrophoresis-based-chemiluminescence-immunoassay-for-the-detection-of-alpha-fetoprotein-in-human-serum
#11
Jingwen Liu, Jingjin Zhao, Shuting Li, Liangliang Zhang, Yong Huang, Shulin Zhao
A sensitive immunoassay method based on microchip electrophoresis chemiluminescence (MCE-CL) detection technology was developed for the detection of tumor marker alpha-fetoprotein (AFP). This method adopts the non-competitive immunoassay mode, and was conducted after AFP reacted with excessive horseradish peroxidase (HRP) labeled monoclonal antibody. The extreme pH value was adopted in the electrophoresis buffer solution. The use of brij 35 as an additive of electrophoresis buffer increased dramatically the resolution (Rs) and the reproducibility of the analysis...
April 1, 2017: Talanta
https://www.readbyqxmd.com/read/28112125/systemic-administration-of-an-anti-tumor-necrosis-factor-alpha-monoclonal-antibody-protects-against-endotoxin-induced-uveitis-in-rats
#12
Qingman Ge, Shaocheng Wang, Yuezhong Zheng
OBJECTIVE: This study was to evaluate the effect of systemic injection of an anti-tumor necrosis factor alpha (TNF-α) monoclonal antibody (mAb) on endotoxin-induced uveitis (EIU). MATERIALS AND METHODS: Fifty-six male Wistar rats (6-8 weeks old) were randomly divided into three groups: EIU, anti-TNF-α mAb + EIU, and control. EIU was induced by injecting Escherichia coli O55:B5 lipopolysaccharide (LPS) into the hind footpad of the rats (150 μg/rat). The anti-TNF-α mAb (1 μg/kg) was administrated 30 min before LPS injection through one-time intravenous injection...
December 2016: Indian Journal of Ophthalmology
https://www.readbyqxmd.com/read/28088282/conjugation-site-analysis-of-antibody-drug-conjugates-adcs-by-signature-ion-fingerprinting-and-normalized-area-quantitation-approach-using-nano-liquid-chromatography-coupled-to-high-resolution-mass-spectrometry
#13
Hua Sang, Gaoyuan Lu, Yazhong Liu, Qiyue Hu, Weifeng Xing, Dongbing Cui, Fang Zhou, Jingwei Zhang, Haiping Hao, Guangji Wang, Hui Ye
Trastuzumab-MCC-DM1 (T-DM1) is an antibody-drug conjugate (ADC) that consists of a monoclonal antibody (mAb) trastuzumab non-cleavably linked to a cytotoxic drug DM1. During production, the DM1 agents were conjugated to the lysine residues of the mAb in a non-specific manner, yielding a heterogeneous mixture of ADC molecules that differ with respect to both the number and the conjugation sites of DM1 per mAb molecule. Since drug conjugation sites of ADC can significantly impact properties such as stability and pharmacokinetic behaviors, a rapid and reliable approach for conjugation site analysis of ADCs is highly demanded...
February 22, 2017: Analytica Chimica Acta
https://www.readbyqxmd.com/read/28087106/charge-variant-analysis-of-proposed-biosimilar-to-trastuzumab
#14
Pravinkumar Dakshinamurthy, Pavithra Mukunda, Bhargav Prasad Kodaganti, Bharath Ravindra Shenoy, Bairavabalakumar Natarajan, Amol Maliwalave, Vivek Halan, Sathyabalan Murugesan, Sunit Maity
Trastuzumab is a humanized monoclonal antibody (mAb) employed for the treatment of HER2 Positive Breast Cancer. A HER2 overexpressing tumor cell binds to Trastuzumab and attracts immune cells which lead to induction of Antibody Dependent Cellular Cytotoxicity (ADCC) by binding to Fc receptors (CD16a or FcγRIIIa) on an effector cell, such as natural killer (NK) cells. The most commonly expressed receptor on NK cell is CD16a which binds to the Fc portion of Trastuzumab. The ligand-independent HER2-HER3 dimerization is the most potent stimulator of downstream pathways for regulation of cell growth and survival...
January 10, 2017: Biologicals: Journal of the International Association of Biological Standardization
https://www.readbyqxmd.com/read/28061947/b-lymphocytes-forming-follicle-like-structures-in-the-lung-tissue-of-tuberculosis-infected-mice-dynamics-phenotypes-and-functional-activity
#15
Irina Linge, Alexander Dyatlov, Elena Kondratieva, Vadim Avdienko, Alexander Apt, Tatiana Kondratieva
During tuberculosis (TB) infection, B cells form follicles in close vicinity of lung granuloma. We assessed the dynamics of follicle formation, surface phenotypes and functional activity of lung B cells during TB course in genetically susceptible mice. The follicles appeared early post infection and peaked at weeks 7-8. Lung B cells resembled classical B2 cells (CD19(+)IgMloIgD(hi)CD1d(-)CD21/35(int)CD5(-)CD11b(-)CD43(-)), but differed from them by the absence of B2 marker CD23. Lung B-cells constitutively expressed MHC II molecules, presented mycobacterial antigens to immune CD4(+) T-cells and produced high amounts of IL-6 and IL-11, but no classical type 1 (TNF-α, IFN-γ), or anti-inflammatory (IL-10, TGF-β) cytokines...
January 2017: Tuberculosis
https://www.readbyqxmd.com/read/28042070/fatigue-during-and-after-breast-cancer-therapy-a-prospective-study
#16
Kristin V Reinertsen, Olav Engebraaten, Jon H Loge, Milada Cvancarova, Bjørn Naume, Erik Wist, Hege Edvardsen, Elisabeth Wille, Trine Bjøro, Cecilie E Kiserud
CONTEXT: Chronic fatigue (CF) in breast cancer (BC) survivors is multifactorial and may be caused by immune activation triggered by BC or its treatment. In the Neoadjuvant Avastin in Breast Cancer study, BC patients received neoadjuvant chemotherapy (FEC100→taxane) ± bevacizumab, a monoclonal antibody with fatigue as a potential side effect. OBJECTIVES: To examine fatigue levels and prevalence of CF before and during chemotherapy and at follow-up, and their associations with C-reactive protein (CRP) and clinical variables...
March 2017: Journal of Pain and Symptom Management
https://www.readbyqxmd.com/read/28008632/in-capillary-approach-to-eliminate-sds-interferences-in-antibody-analysis-by-capillary-electrophoresis-coupled-to-mass-spectrometry
#17
Laura Sánchez-Hernández, Cristina Montealegre, Steffen Kiessig, Bernd Moritz, Christian Neusüß
Capillary electrophoresis is an important technique for the characterization of monoclonal antibodies (mAbs), especially in the pharmaceutical context. However, identification is difficult as upscaling and hyphenation of used methods directly to mass spectrometry is often not possible due to separation medium components that are incompatible with MS detection. Here a CE-MS method for the analysis of mAbs is presented analyzing SDS-complexed samples. To obtain narrow and intensive peaks of SDS-treated antibodies, an in-capillary strategy was developed based on the co-injection of positively charged surfactants and methanol as organic solvent...
December 23, 2016: Electrophoresis
https://www.readbyqxmd.com/read/28000101/remarkable-pharmacokinetics-of-monoclonal-antibodies-a-quest-for-an-explanation
#18
Joannes A A Reijers, Matthijs Moerland, Jacobus Burggraaf
Monoclonal antibodies (MAbs) usually display slow and limited distribution with combined linear and non-linear elimination mechanisms. While studying individual pharmacokinetic profiles, it was noticed that MAb plasma concentration can vary abruptly over time, with one or more increases after the time to maximum plasma concentration when theoretically the concentration should only decline. This article summarizes the frequency of these additional peaks and assesses whether normal intra-subject and assay variability can explain the observations...
December 20, 2016: Clinical Pharmacokinetics
https://www.readbyqxmd.com/read/27997765/intracellular-cho-cell-metabolite-profiling-reveals-steady-state-dependent-metabolic-fingerprints-in-perfusion-culture
#19
Daniel J Karst, Robert Steinhoff, Marie R G Kopp, Elisa Serra, Miroslav Soos, Renato Zenobi, Massimo Morbidelli
Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors...
December 20, 2016: Biotechnology Progress
https://www.readbyqxmd.com/read/27994435/pkr-promotes-choroidal-neovascularization-via-upregulating-the-pi3k-akt-signaling-pathway-in-vegf-expression
#20
Manhui Zhu, Xiaojuan Liu, Shengcun Wang, Jin Miao, Liucheng Wu, Xiaowei Yang, Ying Wang, Lihua Kang, Wendie Li, Chen Cui, Hui Chen, Aimin Sang
PURPOSE: The aim of this study was to investigate the functions of dsRNA-activated protein kinase (PKR) in choroidal neovascularization (CNV) and related signaling pathways in the production of vascular endothelial growth factor (VEGF). METHODS: A chemical hypoxia model of in vitro RF/6A cells, a rhesus choroid-retinal endothelial cell line, was established by adding cobalt chloride (CoCl2) to the culture medium. PKR, phosphophosphatidylinositol 3-kinase (p-PI3K), phosphoprotein kinase B (p-Akt), and VEGF protein levels in RF/6A cells were detected with western blotting...
2016: Molecular Vision
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