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https://www.readbyqxmd.com/read/29761239/kinetics-and-characterization-of-non-enzymatic-fragmentation-of-monoclonal-antibody-therapeutics
#1
Sahithi Ravuluri, Rohit Bansal, Nidhi Chhabra, Anurag S Rathore
PURPOSE: To understand non-enzymatic hydrolytic fragmentation of a monoclonal antibody therapeutic under temperature stressed conditions and investigating possible mechanism for the same. METHODS: The mAb therapeutic was incubated at 50°C in phosphate buffer at pH 6.5 and fragmentation was monitored at different ionic strengths under stressed conditions. The incubated mAb was sampled at regular time intervals by analytical Size Exclusion Chromatography (SEC). RESULTS: It was observed that 57% of the mAb product fragmented over 4 days into two fragment species - Fc-Fab and Fab with molecular weights of 97 KDa and 47 KDa, respectively, as measured by mass spectrometry (MS) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)...
May 14, 2018: Pharmaceutical Research
https://www.readbyqxmd.com/read/29746518/an-ambient-temperature-stable-antitoxin-of-nine-co-formulated-antibodies-for-botulism-caused-by-serotypes-a-b-and-e
#2
Mingxiang Li, Dennis Lee, Chidi R Obi, Joel K Freeberg, Shauna Farr-Jones, Milan T Tomic
Safe and effective antitoxins to treat and prevent botulism are needed for biodefense. We have developed recombinant antibody-based therapeutics for botulinum neurotoxin (BoNT) serotypes A, B, and E. The mechanism of action of this antitoxin requires that three mAbs bind one toxin molecule to achieve clearance. Here we present a co-formulation of an antitoxin to the three most important serotypes. Combining these antibodies obviates the need to identify the serotype causing intoxication prior to drug administration, which would facilitate administration...
2018: PloS One
https://www.readbyqxmd.com/read/29723667/the-missing-piece-in-the-puzzle-prediction-of-aggregation-via-the-protein-protein-interaction-parameter-a-%C3%A2-2
#3
Ellen Koepf, Rudolf Schroeder, Gerald Brezesinski, Wolfgang Friess
The tendency of protein pharmaceuticals to form aggregates is a major challenge during formulation development, as aggregation affects quality and safety of the product. In particular, the formation of large native-like particles in the context of liquid-air interfacial stress is a well-known but not fully understood problem. Focusing on the two most fundamental criteria of protein formulation affecting protein-protein interaction, the impact of pH and ionic strength on the interaction parameter A∗ 2 and its link to aggregation upon mechanical stress was investigated...
April 30, 2018: European Journal of Pharmaceutics and Biopharmaceutics
https://www.readbyqxmd.com/read/29679468/high-efficiency-affinity-precipitation-of-multiple-industrial-mabs-and-fc-fusion-proteins-from-cell-culture-harvests-using-z-elp-e2-nanocages
#4
Andrew R Swartz, Xuankuo Xu, Steven J Traylor, Zheng Jian Li, Wilfred Chen
Affinity precipitation using Z-ELP-E2 nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high-yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multi-valent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A-D) and one Fc fusion protein (Fc A) with diverse molecular properties...
April 21, 2018: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/29665321/a-novel-ano5-splicing-variant-in-a-lgmd2l-patient-leads-to-production-of-a-truncated-aggregation-prone-ano5-peptide
#5
Jing Xu, Li Xu, Yeh S Lau, Yandi Gao, Steven A Moore, Renzhi Han
Mutations in ANO5 cause several human diseases including gnathodiaphyseal dysplasia 1 (GDD1), limb-girdle muscular dystrophy 2L (LGMD2L), and Miyoshi myopathy 3 (MMD3). Previous work showed that complete genetic disruption of Ano5 in mice did not recapitulate human muscular dystrophy, while residual expression of mutant Ano5 in a gene trapped mouse developed muscular dystrophy with defective membrane repair. This suggests that truncated Ano5 expression may be pathogenic. Here, we screened a panel of commercial anti-Ano5 antibodies using a recombinant adenovirus expressing human Ano5 with FLAG and YFP at the N- and C-terminus, respectively...
April 2018: Journal of Pathology. Clinical Research
https://www.readbyqxmd.com/read/29662239/elimination-of-tdp-43-inclusions-linked-to-amyotrophic-lateral-sclerosis-by-a-misfolding-specific-intrabody-with-dual-proteolytic-signals
#6
Yoshitaka Tamaki, Akemi Shodai, Toshifumi Morimura, Ryota Hikiami, Sumio Minamiyama, Takashi Ayaki, Ikuo Tooyama, Yoshiaki Furukawa, Ryosuke Takahashi, Makoto Urushitani
Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is implicated in the pathogenesis of sporadic and certain familial forms of amyotrophic lateral sclerosis (ALS), suggesting elimination of TDP-43 aggregates as a possible therapeutic strategy. Here we generated and investigated a single-chain variable fragment (scFv) derived from the 3B12A monoclonal antibody (MAb) that recognises D247 of the TDP-43 nuclear export signal, an epitope masked in the physiological state. In transfected HEK293A cells, 3B12A scFv recapitulated the affinity of the full-length MAb to mislocalised TDP-43 with a defective nuclear localising signal and to a TDP-43 inclusion mimic with cysteine-to-serine substitution at RRM1...
April 16, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29655788/antibody-engineering-to-improve-manufacturability
#7
Sujeewa D Wijesuriya, Elizabeth Pongo, Milan Tomic, Fangjiu Zhang, Consuelo Garcia-Rodriquez, Fraser Conrad, Shauna Farr-Jones, James D Marks, Arnold H Horwitz
Expression variation among antibodies produced by stably transfected Chinese Hamster Ovary (CHO) cells is well established. While developing CHO-K1 cell lines, we encountered a human monoclonal antibody, mAb B-c, with severe manufacturability issues, including very poor expression and high levels of heavy chain (HC) dimer and high molecular weight aggregates. Using transient expression in CHO-K1 cells, we identified light chain (LC) as the source of the manufacturability issues for this antibody. While other antibodies achieved optimal expression at 1:1 or 2:1 LC to HC ratios, mAb B-c required up to a 6:1 LC:HC for maximal expression, which was still significantly lower than that for other tested antibodies...
April 12, 2018: Protein Expression and Purification
https://www.readbyqxmd.com/read/29648589/analysis-of-the-3h8-antigen-of-candidaalbicans-reveals-new-aspects-of-the-organization-of-fungal-cell-wall-proteins
#8
Rafael Sentandreu, Antonio Caminero, Itzel Rentería, Claudia León-Ramirez, Luis González-de-la-Vara, Eulogio Valentin-Gomez, José Ruiz-Herrera
The walls of both, yeast and mycelial cells of Candida albicans possess a species specific antigen that is recognized by a monoclonal antibody (MAb 3H8). This antigen can be extracted in the form of a very high Mr complex, close or over 106 Da, by treatment, with β-1,3 glucanase, β mercaptoethanol or dithothreitol, or mild alkali, but not by saturated HF in pyridine, suggesting that the complex is bound to wall β-1,3 glucans, and to proteins by disulfide bonds, but not to β-1,6 glucans. Through its sensitivity to trypsin and different deglycosylation procedures, it was concluded that the epitope is associated to a glycoprotein containing N-glycosidic, but not O-glycosidic mannan moieties...
April 10, 2018: FEMS Yeast Research
https://www.readbyqxmd.com/read/29626535/biophysical-properties-and-heating-induced-aggregation-of-lysine-conjugated-antibody-drug-conjugates
#9
Aditya V Gandhi, Keith J Arlotta, Hsiao-Nung Chen, Shawn C Owen, John F Carpenter
The commercially available antibody-drug conjugate (ADC) product, Kadcyla® is synthesized using a two-step reaction, wherein the linker is conjugated to native lysines on the monoclonal antibody (mAb) in step 1, followed by drug conjugation to the linker-modified antibody in step 2. In our study, we synthesized a lysine conjugated ADC (Syn-ADC) on the same trastuzumab scaffold as Kadcyla® using a one-step reaction. Mass spectrometry of both products revealed a sub-population of Kadcyla® containing free linkers conjugated to the mAb, but not conjugated to the drug, which were absent in the one-step reaction ADC product...
April 4, 2018: Journal of Pharmaceutical Sciences
https://www.readbyqxmd.com/read/29625180/a%C3%AE-oligomer-uptake-and-the-resulting-inflammatory-response-in-adult-human-astrocytes-are-precluded-by-an-anti-a%C3%AE-single-chain-variable-fragment-in-combination-with-an-apoe-mimetic-peptide
#10
Laia Montoliu-Gaya, Sandra D Mulder, Maaike A C Herrebout, Johannes C Baayen, Sandra Villegas, Robert Veerhuis
An imbalance between production and clearance of soluble amyloid-β (Aβ) initiates the pathological process in sporadic Alzheimer's disease (AD). Aβ-specific antibodies seemed promising as therapeutic option in AD mouse models. In patients, however, vascular side-effects and Aβ-antibody complex-induced microglial and/or perivascular macrophage inflammatory responses were encountered. To prevent inflammatory reactions, we designed a single chain variable fragment (scFv-h3D6), based on monoclonal antibody bapineuzumab (mAb-h3D6), but lacking the Fc region...
April 3, 2018: Molecular and Cellular Neurosciences
https://www.readbyqxmd.com/read/29622226/specific-colorimetric-elisa-method-based-on-dna-hybridization-reaction-and-non-crosslinking-gold-nanoparticles-aggregation-for-the-detection-of-amantadine
#11
Fang-Fei Zhu, Juan Peng, Zhen Huang, Li-Ming Hu, Gang-Gang Zhang, Dao-Feng Liu, Ke-Yu Xing, Kai-Yi Zhang, Wei-Hua Lai
Amantadine (AMD), a banned antiviral veterinary drug, is still being abused. This study developed a novel enzyme linked immunosorbent assay for the colorimetric detection of AMD involving DNA hybridization reaction and non-crosslinking gold nanoparticles (AuNPs) aggregation. Accordingly, the Primer 1-AuNPs-anti-AMD monoclonal antibody (mAb) could be captured by AMD artificial antigen on ELISA wells. Primer 2, which was complementary paired to Primer 1, was eventually added into the ELISA wells. After the hybridization reaction, the free Primer 2 in the supernatant was mixed with AuNPs and NaCl and induced a rapid color change of AuNPs...
August 15, 2018: Food Chemistry
https://www.readbyqxmd.com/read/29600470/photokinetic-drug-delivery-near-infrared-nir-induced-permeation-enhancement-of-bevacizumab-ranibizumab-and-aflibercept-through-human-sclera
#12
Steven A Giannos, Edward R Kraft, Zhen-Yang Zhao, Kevin H Merkley, Jiyang Cai
PURPOSE: Permeation studies, with near infrared (NIR) light and anti-aggregation antibody formulation, were used to investigate the in vitro permeation of bevacizumab, ranibizumab and aflibercept through human sclera. METHODS: A vertical, spherical Franz cell diffusion apparatus was used for this scleral tissue permeation model. A photokinetic ocular drug delivery (PODD) testing device accommodated the placement of NIR LEDs above the donor chambers. An adjustable LED driver/square wave generator provided electrical energy with a variable pulse rate and pulse width modulation (duty cycle)...
March 29, 2018: Pharmaceutical Research
https://www.readbyqxmd.com/read/29556730/impact-of-buffer-protein-concentration-and-sucrose-addition-on-the-aggregation-and-particle-formation-during-freezing-and-thawing
#13
Astrid Hauptmann, Katja Podgoršek, Drago Kuzman, Stanko Srčič, Georg Hoelzl, Thomas Loerting
PURPOSE: This study addresses the effect of freezing and thawing on a therapeutic monoclonal antibody (mAb) solution and the corresponding buffer formulation. Particle formation, crystallization behaviour, morphology changes and cryo-concentration effects were studied after varying the freezing and thawing rates, buffer formulation and protein concentration. The impact of undergoing multiple freeze/thaw (FT)-cycles at controlled and uncontrolled temperature rates on mAb solutions was investigated in terms of particle formation...
March 19, 2018: Pharmaceutical Research
https://www.readbyqxmd.com/read/29551237/chemical-modification-of-protein-a-chromatography-ligands-with-polyethylene-glycol-ii-effects-on-resin-robustness-and-process-selectivity
#14
Justin Weinberg, Shaojie Zhang, Allison Kirkby, Enosh Shachar, Giorgio Carta, Todd Przybycien
We have proposed chemical modification of Protein A (ProA) chromatography ligands with polyethylene glycol (PEGylation) as a strategy to increase the resin selectivity and robustness by providing the ligand with a steric repulsion barrier against non-specific binding. Here, we report on robustness and selectivity benefits for Repligen CaptivA PriMAB resin with ligands modified with 5.2 kDa and 21.5 kDa PEG chains, respectively. PEGylation of ProA ligands allowed the resin to retain a higher percentage of static binding capacity relative to the unmodified resin upon digestion with chymotrypsin, a representative serine protease...
February 24, 2018: Journal of Chromatography. A
https://www.readbyqxmd.com/read/29540956/evaluation-of-fluorescent-dyes-to-measure-protein-aggregation-within-mammalian-cell-culture-supernatants
#15
Sheun Oshinbolu, Rachana Shah, Gary Finka, Mike Molloy, Mark Uden, Daniel G Bracewell
BACKGROUND: A current challenge in bioprocessing is the ability to analyse critical quality attributes such as aggregation without prior purification. This study evaluated the use of fluorescent dyes (Bis-ANS, SYPRO Orange, Thioflavin T and ProteoStat) to characterise mAb aggregates in Chinese hamster ovary clarified cultures. RESULTS: The null and mAb culture supernatants showed an increase in fluorescence intensity over the duration of the culture. The null cultures on day 14 saw a rapid increase in fluorescence intensity; day 10 to day 14, Bis-ANS and Thioflavin T had average increases of 21% and 48%, respectively, whereas ProteoStat and SYPRO Orange showed an average increase of 60%...
March 2018: Journal of Chemical Technology and Biotechnology
https://www.readbyqxmd.com/read/29522957/protein-a-affinity-chromatography-of-chinese-hamster-ovary-cho-cell-culture-broths-containing-biopharmaceutical-monoclonal-antibody-mab-experiments-and-mechanistic-transport-binding-and-equilibrium-modeling
#16
Matic Grom, Mirijam Kozorog, Simon Caserman, Andrej Pohar, Blaž Likozar
Protein A-based affinity chromatography is a highly-efficient separation method to capture, purify and isolate biosimilar monoclonal antibodies (mAb) - an important medical product of biopharmaceutical industrial manufacturing. It is considered the most expensive step in purification downstream operations; therefore, its performance optimization offers a great cost saving in the overall production expenditure. The biochemical mixture-separating specific interaction experiments with Chinese hamster ovary (CHO) cell culture harvest, containing glycosylated extracellular immunoglobulins (Ig), were made using five different state-of-the-art commercial resins...
April 15, 2018: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
https://www.readbyqxmd.com/read/29492680/formulation-stabilization-and-disaggregation-of-bevacizumab-ranibizumab-and-aflibercept-in-dilute-solutions
#17
Steven A Giannos, Edward R Kraft, Zhen-Yang Zhao, Kevin H Merkley, Jiyang Cai
PURPOSE: Studies were conducted to investigate dilute solutions of the monoclonal antibody (mAb) bevacizumab, mAb fragment ranibizumab and fusion protein aflibercept, develop common procedures for formulation of low concentration mAbs and identify a stabilizing formulation for anti-VEGF mAbs for use in in vitro permeation studies. METHODS: Excipient substitutions were screened. The most stabilizing formulation was chosen. Standard dilutions of bevacizumab, ranibizumab and aflibercept were prepared in PBS, manufacturer's formulation, and the new formulation...
February 28, 2018: Pharmaceutical Research
https://www.readbyqxmd.com/read/29482157/il-17a-promotes-the-formation-of-deep-vein-thrombosis-in-a-mouse-model
#18
Peiwu Ding, Shaoshao Zhang, Miao Yu, Yuqian Feng, Qi Long, Huimin Yang, Jingdong Li, Min Wang
Deep venous thrombosis (DVT) is a significant problem in the health care industry worldwide. However, the factors and signaling pathways that trigger DVT formation are still largely unknown. In this study, we investigated the role of interleukin-17A (IL-17A) in DVT formation, focusing on the role of platelet aggregation, neutrophil infiltration, and endothelium cell (EC) activation. Notably, IL-17A levels increased in DVT patients as well as in a mouse DVT model. The DVT model mice were injected with recombinant mouse-IL-17A (rIL-17A) or anti-IL-17A monoclonal antibody (mAb) to further evaluate the effects of this cytokine...
April 2018: International Immunopharmacology
https://www.readbyqxmd.com/read/29446917/mapping-the-mab-aggregation-propensity-using-self-interaction-chromatography-as-a-screening-tool
#19
Sarah H M Hedberg, DongKyu Lee, Yash Mishra, Jonathan M Haigh, Daryl R Williams
The osmotic second virial coefficient ( B2 ), which describes protein-protein molecular interactions in solution, was determined using self-interaction chromatography (SIC) for an IgG1-type mAb across a wide range of solution conditions. These data were compared with its time dependent aggregation behavior, as determined using size-exclusion chromatography (SEC), and its temperature dependent aggregation behavior using dynamic light scattering (DLS) over a four-week period (SEC) or overnight (DLS). DLS and SEC gave consistent data on aggregation behavior, which correlated well with experimental B2 trends across the wide pH (4-9) and NaCl concentration (0-1...
March 20, 2018: Analytical Chemistry
https://www.readbyqxmd.com/read/29425047/protein-adsorption-and-layer-formation-at-the-stainless-steel-solution-interface-mediates-shear-induced-particle-formation-for-an-igg1-monoclonal-antibody
#20
Cavan K Kalonia, Frank Heinrich, Joseph E Curtis, Sid Raman, Maria A Miller, Steven D Hudson
Passage of specific protein solutions through certain pumps, tubing, and/or filling nozzles can result in the production of unwanted subvisible protein particles (SVPs). In this work, surface-mediated SVP formation was investigated. Specifically, the effects of different solid interface materials, interfacial shear rates, and protein concentrations on SVP formation were measured for the National Institute of Standards and Technology monoclonal antibody (NISTmAb), a reference IgG1 monoclonal antibody (mAb). A stainless steel rotary piston pump was used to identify formulation and process parameters that affect aggregation, and a flow cell (alumina or stainless steel interface) was used to further investigate the effect of different interface materials and/or interfacial shear rates...
March 5, 2018: Molecular Pharmaceutics
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