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Antibody purification

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https://www.readbyqxmd.com/read/28728084/purification-of-monoclonal-antibodies-entirely-in-flow-through-mode
#1
Tsuyoshi Yamada, Koichi Yamamoto, Takashi Ishihara, Shigeru Ohta
A depth filter is widely used in biopharmaceutical manufacturing process. In this study, we found that buffer exchange to reduce conductivity dramatically improved the removal of impurities in depth filtration. The host cell protein clearance was comparable to that of protein A affinity chromatography, which is generally used as a first capture step in monoclonal antibody purification. In addition, a combination of different depth filters showed enhanced purification. Additional flow-through purification is possible without any sample preparation, and a biopharmaceutical-quality purity level (<100ppm for host cell protein and <5% for high molecular weight species) can be attained...
July 3, 2017: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/28726418/quantitative-assessment-of-the-effects-of-trypsin-digestion-methods-on-affinity-purification-mass-spectrometry-based-protein-protein-interaction-analysis
#2
Yueqing Zhang, Hong Sun, Jing Zhang, Allan R Brasier, Yingxin Zhao
Affinity purification-mass spectrometry (AP-MS) has become the method of choice for discovering protein-protein interactions (PPIs) under native conditions. The success of AP-MS depends on the efficiency of trypsin digestion and the recovery of the tryptic peptides for MS analysis. Several different protocols have been used for trypsin digestion of protein complexes in AP-MS studies, but no systematic studies have been conducted on the impact of trypsin digestion conditions on the identification of PPIs. Here, we used NFκB/RelA and Bromodomain-containing protein 4 (BRD4) as baits and test five distinct trypsin digestion methods (two using "on-beads," three using "elution-digestion" protocols)...
July 20, 2017: Journal of Proteome Research
https://www.readbyqxmd.com/read/28722325/polymer-mediated-flocculation-of-transient-cho-cultures-as-a-simple-high-throughput-method-to-facilitate-antibody-discovery
#3
Matthew G Schmitt, Yashas Rajendra, Maria D Hougland, Jeffrey S Boyles, Gavin C Barnard
Most biopharmaceutical drugs, especially monoclonal antibodies (mAbs), bispecific antibodies (BsAbs) and Fc-fusion proteins, are expressed using Chinese Hamster Ovary (CHO) cell lines. CHO cells typically yield high product titers and high product quality. Unfortunately, CHO cell lines also generate high molecular weight (HMW) aggregates of the desired product during cell culture along with CHO host cell protein (HCP) and CHO DNA. These immunogenic species, co-purified during Protein A purification, must be removed in a multi-step purification process...
July 19, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/28720505/chemically-generated-igg2-bispecific-antibodies-through-disulfide-bridging
#4
James T Patterson, Edwige Gros, Heyue Zhou, Ghazi Atassi, Lisa Kerwin, Lisa Carmody, Tong Zhu, Bryan Jones, Yanwen Fu, Gunnar F Kaufmann
Bispecific antibodies (BsAbs) are designed to engage two antigens simultaneously, thus, effectively expanding the ability of antibody-based therapeutics to target multiple pathways within the same cell, engage two separate soluble antigens, bind the same antigen with distinct paratopes, or crosslink two different cell types. Many recombinant BsAb formats have emerged, however, expression and purification of such constructs can often be challenging. To this end, we have developed a chemical strategy for generating BsAbs using native IgG2 architecture...
July 8, 2017: Bioorganic & Medicinal Chemistry Letters
https://www.readbyqxmd.com/read/28720222/molecular-insight-into-protein-binding-orientations-and-interaction-modes-on-hydrophobic-charge-induction-resin
#5
Hong-Fei Tong, Carlo Cavallotti, Shan-Jing Yao, Dong-Qiang Lin
Hydrophobic charge-induction chromatography (HCIC) with 4-mercaptoethyl-pyridine (MEP) as the functional ligand has been developed as a new technology for antibody purification. In the present work, molecular simulation methods were developed to investigate the interactions between the Fc fragment of IgG and a MEP ligand net. The MM/PBSA method was used to evaluate the binding energy for the MEP ligand net at different densities. It was found that ligand density had significant influence on the binding of Fc...
June 30, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28713027/an-on-matrix-digestion-procedure-for-ap-ms-experiments-dissects-the-interplay-between-complex-conserved-and-serotype-specific-reactivities-in-dengue-virus-human-plasma-interactome
#6
Yassel Ramos, Vivian Huerta, Dayron Martín, Sucel Palomares, Alexis Yero, Dianne Pupo, Sebastien Gallien, Alejandro M Martín, Yasset Pérez-Riverol, Mónica Sarría, Osmany Guirola, Glay Chinea, Bruno Domon, Luis Javier González
The interactions between the four Dengue virus (DENV) serotypes and plasma proteins are crucial in the initial steps of viral infection to humans. Affinity purification combined with quantitative mass spectrometry analysis, has become one of the most powerful tools for the investigation on novel protein-protein interactions. Using this approach, we report here that a significant number of bait-interacting proteins do not dissociate under standard elution conditions, i.e. acid pH and chaotropic agents, and that this problem can be circumvented by using the "on-matrix" digestion procedure described here...
July 13, 2017: Journal of Proteomics
https://www.readbyqxmd.com/read/28710478/recombinant-fasciola-hepatica-fatty-acid-binding-protein-suppresses-toll-like-receptor-stimulation-in-response-to-multiple-bacterial-ligands
#7
Marcos J Ramos-Benítez, Caleb Ruiz-Jiménez, Vasti Aguayo, Ana M Espino
Recently, we reported that a native Fasciola hepatica fatty acid binding protein (FABP) termed Fh12 is a powerful anti-inflammatory protein capable of suppressing the LPS-induced expression of inflammatory markers in vivo and in vitro. Because the purification of a protein in native form is, in many situations not cost-beneficial and unsuitable for industrial grade scale-up, this study accomplished the task of optimizing the expression and purification of a recombinant form of FABP (Fh15). Additionally, we ascertained whether this molecule could exhibit a similar suppressive effect on TLR-stimulation and inflammatory cytokine expression from macrophages than those previously demonstrated for the native molecule...
July 14, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28708446/elisa-reagent-coverage-evaluation-by-affinity-purification-tandem-mass-spectrometry
#8
Scott M Henry, Elissa Sutlief, Oscar Salas-Solano, John Valliere-Douglass
Host cell proteins (HCPs) must be adequately removed from recombinant therapeutics by downstream processing to ensure patient safety, product quality, and regulatory compliance. HCP process clearance is typically monitored by enzyme-linked immunosorbent assay (ELISA) using a polyclonal reagent. Recently, mass spectrometry (MS) has been utilized to identify specific HCP process impurities and monitor their clearance. Despite this capability, ELISA remains the preferred analytical approach due to its simplicity and throughput...
July 14, 2017: MAbs
https://www.readbyqxmd.com/read/28706572/a-microwell-printing-fabrication-strategy-for-the-on-chip-templated-biosynthesis-of-protein-microarrays-for-surface-plasmon-resonance-imaging
#9
Gerald Manuel, Andrej Lupták, Robert M Corn
A two-step templated, ribosomal biosynthesis/printing method for the fabrication of protein microarrays for surface plasmon resonance imaging (SPRI) measurements is demonstrated. In the first step, a sixteen component microarray of proteins is created in microwells by cell free on chip protein synthesis; each microwell contains both an in vitro transcription and translation (IVTT) solution and 350 femtomoles of a specific DNA template sequence that together are used to create approximately 40 picomoles of a specific hexahistidine-tagged protein...
September 22, 2016: Journal of Physical Chemistry. C, Nanomaterials and Interfaces
https://www.readbyqxmd.com/read/28706253/affinity-purification-of-erythropoietin-from-cell-culture-supernatant-combined-with-maldi-tof-ms-analysis-of-erythropoietin-n-glycosylation
#10
David Falck, Markus Haberger, Rosina Plomp, Michaela Hook, Patrick Bulau, Manfred Wuhrer, Dietmar Reusch
Erythropoietin (EPO) is a heavily glycosylated hormone whose recombinant forms are used for treatment of anaemia. EPO glycosylation is important for its pharmacological properties. An analytical workflow, which can determine EPO glycosylation in an accurate and high-throughput fashion from cell culture supernatant (CCS) in approximately 24 h, offers the possibility to follow changes during production. To address this challenge, we present a complete workflow consisting of protein purification, glycan release, sialic acid derivatization, solid phase extraction, matrix-assisted laser desorption/ionization - mass spectrometry (MALDI-MS) analysis and MassyTools data processing...
July 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28697990/avian-antibodies-igy-against-trypanosoma-cruzi-purification-and-characterization-studies
#11
Thirssa H Grando, Matheus D Baldissera, Mariângela F de Sá, Guilherme do Carmo, Bianca Carolina Z Porto, Gisele S V Aguirre, Maria Isabel de Azevedo, Francielli P K de Jesus, Janio M Santurio, Michele R Sagrillo, Lenita Moura Stefani, Silvia Gonzalez Monteiro
Trypanosoma cruzi is a flagellated protozoan belonging to the Trypanosomatidae family, the etiologic agent of Chagas disease. Currently, there is neither a licensed vaccine nor effective treatment, characterizing an unmet clinical need. The IgY refers to the egg yolk immunoglobulin (Y=yolk) and its production and use are subjects of many studies due to the diversity of its diagnostic and therapeutic applications. Several researchers have shown that the use of specific IgY may prevent and/or control infectious and parasitic diseases...
July 8, 2017: Journal of Immunological Methods
https://www.readbyqxmd.com/read/28697935/continuous-countercurrent-tangential-chromatography-for-mixed-mode-post-capture-operations-in-monoclonal-antibody-purification
#12
Amit K Dutta, Dmitriy Fedorenko, Jasmine Tan, Joseph A Costanzo, David S Kahn, Andrew L Zydney, Oleg Shinkazh
Continuous Countercurrent Tangential Chromatography (CCTC) has been shown to demonstrate significant advantages over column chromatography including higher productivity, lower operational pressure, disposable flow path, and lower resin use. Previous applications of CCTC have been limited to initial capture of monoclonal antibodies (mAb) from clarified cell culture harvest. In this present article, a CCTC system was designed and tested for a post-capture antibody purification step. Mixed mode cation exchange-hydrophobic interaction chromatography resins with two different particle sizes were used to reduce host cell protein (HCP), leached protein A, DNA, and aggregates from a mAb stream after a protein A operation...
June 22, 2017: Journal of Chromatography. A
https://www.readbyqxmd.com/read/28696676/alternative-affinity-ligands-for-immunoglobulins
#13
Nika Kruljec, Tomaž Bratkovič
The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. Especially the development of better affinity chromatography matrices, supporting robust, time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene...
July 11, 2017: Bioconjugate Chemistry
https://www.readbyqxmd.com/read/28684274/construction-of-high-level-prokaryotic-expression-and-purification-system-of-pd-l1-extracellular-domain-by-using-escherichia-coli-host-cell-machinery
#14
Muhammad Kalim, Jie Chen, Shenghao Wang, Caiyao Lin, Saif Ullah, Keying Liang, Qian Ding, Shuqing Chen, Jinbiao Zhan
Programmed cell death 1 ligand 1 (PD-L1) is a trans-membrane protein highly expressed on the membrane of cancer cell, which binds inhibitory receptor of PD-1 on the T cells and attenuates anti-tumor immune response.The strategy of blocking PD1 and PD-L1 interaction has been widely used for anti-cancer drug development. The DNA encoding extracellular domain of PD-L1wascloned and expressed with the pET30(+) and Escherichia coli BL21(DE3) system. Cloning of PD-L1 extracellular domain was confirmed by PCR and enzymatic digestion...
July 3, 2017: Immunology Letters
https://www.readbyqxmd.com/read/28679315/bone-marrow-mesenchymal-stem-cell-derived-cd63-exosomes-transport-wnt3a-exteriorly-and-enhance-dermal-fibroblast-proliferation-migration-and-angiogenesis-in-vitro
#15
Jeffrey D McBride, Luis Menocal-Rodriguez, Ambar Candanedo, Wellington Guzman, Marta Garcia-Contreras, Evangelos Van Badiavas
Wnts are secreted glycoproteins that regulate stem cell self-renewal, differentiation, and cell-to-cell communication during embryonic development and in adult tissues. Bone marrow mesenchymal stem cells (BM-MSCs) have been shown to stimulate dermis repair and regeneration; however, it is unclear how BM-MSCs may modulate downstream Wnt signaling. While recent reports implicate that Wnt ligands and Wnt messenger RNAs (such as Wnt4) exist within the interior compartment of exosomes, it has been debated whether or not Wnts exist on the exterior surface of exosomes to travel in the extracellular space...
July 5, 2017: Stem Cells and Development
https://www.readbyqxmd.com/read/28677761/expression-and-purification-of-a-major-allergen-pla-a-1-from-platanus-acerifolia-pollen-and-the-preparation-of-its-monoclonal-antibody
#16
Wei-Wei Ni, Wen Huang, De-Qin Wu, Yan-Jun Zhou, Chun-Mei Ji, Meng-Da Cao, Miao Guo, Jin-Lu Sun, Ji-Fu Wei
Platanus acerifolia pollen is considered an important source of airborne allergens in numerous cities. Pla a 1 is a major allergen from P. acerifolia pollen. The present study aimed to express and purify Pla a 1, and to prepare its monoclonal antibody. In the present study, the Pla a 1 gene was subcloned into a pET‑28a vector and transformed into the ArcticExpress™ (DE3) RP Escherichia coli host strain. The purified Pla a 1 was then used to immunize BALB/c mice. When serum detection was positive, spleen cells were isolated from the mice and fused with SP2/0 myeloma cells at a ratio of 10:1...
June 30, 2017: Molecular Medicine Reports
https://www.readbyqxmd.com/read/28668757/determination-of-longr-3-igf-i-r-3-igf-i-des1-3-igf-i-and-their-metabolites-in-human-plasma-samples-by-means-of-lc-ms
#17
Andreas Thomas, Katja Walpurgis, Philippe Delahaut, Eric Fichant, Wilhelm Schänzer, Mario Thevis
According to the regulations of the World Anti-Doping Agency (WADA), growth promoting peptides such as the insulin-like growth factor-I (IGF-I) and its synthetic analogues belong to the class of prohibited compounds. While several assays to quantify endogenous IGF-I have been established, the potential misuse of synthetic analogues such as LongR(3)-IGF-I, R(3)-IGF-I and Des1-3-IGF-I remains a challenge and superior pharmacokinetic properties have been described for these analogues. Within the present study, it was demonstrated that the target peptides can be successfully detected in plasma samples by means of magnetic beads-based immunoaffinity purification and subsequent nanoscale liquid chromatographic separation with high resolution mass spectrometric detection...
June 27, 2017: Growth Hormone & IGF Research
https://www.readbyqxmd.com/read/28667528/purification-of-natural-antibodies-against-tau-protein-by-affinity-chromatography
#18
Michala Krestova, Lenka Hromadkova, Jan Ricny
Natural antibodies are now widely studied for their therapeutical potential. Therefore, their isolation and subsequent characterization is desired. Here, we describe an isolation method for natural anti-tau antibodies from human plasma by utilization of affinity chromatography with epoxy-activated copolymer resin. The evalution of isolation efficiency and avidity of isolated antibodies is decribed by modified indirect ELISA assay.
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28666898/overexpression-and-characterization-of-the-100k-protein-of-fowl-adenovirus-4-as-an-antiviral-target
#19
Majid Ali Shah, Raheem Ullah, Matteo De March, Muhammad Salahuddin Shah, Fouzia Ismat, Mudasser Habib, Mazhar Iqbal, Silvia Onesti, Moazur Rahman
100K is an important scaffolding protein of adenoviruses including fowl adenovirus serotype 4 (FAdV-4) that causes inclusion body hepatitis-hydropericardium syndrome (IBH-HPS) in poultry. 100K carries out the trimerization of the major capsid hexon protein of the virus for the generation of new virions inside the target host cells. Despite its critical role for FAdV-4, no structural study, in particular, has been conducted so far. Here, the overexpression of soluble 100K protein was successfully carried out in E...
June 28, 2017: Virus Research
https://www.readbyqxmd.com/read/28662655/identification-characterization-and-purification-of-porcine-quiescin-q6-sulfydryl-oxidase-2-protein
#20
Yu-Wen Kuo, Radhika Joshi, Tse-En Wang, Hui-Wen Chang, Sheng-Hsiang Li, Chun-Ni Hsiao, Pei-Shiue Jason Tsai
BACKGROUND: Post-spermiogenesis membrane surface modifications rely on molecules present in the reproductive tracts. Two isoforms (isoform 1 and 2) from Quiescin Q6-Sulfydryl Oxidase protein family have been identified in the male reproductive tract of rodent species. However, unlike isoform 1, scarce information is available for isoform 2, likely due to its lower expression level and lack of proper purification methods to obtain sufficient protein quantity for further assays. RESULTS: This study demonstrated the presence of short and long forms of Quiescin Q6-Sulfydryl Oxidase 2 in boar, likely representing the secretory (short form) and transmembrane (long form) forms of Quiescin Q6-Sulfydryl Oxidase 2...
June 29, 2017: BMC Veterinary Research
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