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https://www.readbyqxmd.com/read/28639328/immunogenicity-of-protein-aggregates-of-a-monoclonal-antibody-generated-by-forced-shaking-stress-with-siliconized-and-nonsiliconized-syringes-in-balb-c-mice
#1
Tomonobu Uchino, Yasunori Miyazaki, Takuto Yamazaki, Yoshiyuki Kagawa
OBJECTIVE: In this study, we aimed to investigate the immunogenicity of protein aggregates of monoclonal antibodies (mAbs), generated by forced shaking stress with siliconized and nonsiliconized syringes in a mouse model. METHODS: Samples were filled in siliconized and nonsiliconized syringes with shaking and headspace air. Characterization studies were performed using high-performance size-exclusion chromatography, nanoparticle tracking analysis, flow cytometry, micro-flow imaging and resonant mass measurement...
June 21, 2017: Journal of Pharmacy and Pharmacology
https://www.readbyqxmd.com/read/28633643/enhancement-of-thermoalkaliphilic-xylanase-production-by-pichia-pastoris-through-novel-fed-batch-strategy-in-high-cell-density-fermentation
#2
Tingting Shang, Dayong Si, Dongyan Zhang, Xuhui Liu, Longmei Zhao, Cong Hu, Yu Fu, Rijun Zhang
BACKGROUND: Xylanase degrades xylan into monomers of various sizes by catalyzing the endohydrolysis of the 1,4-β-D-xylosidic linkage randomly, possessing potential in wide industrial applications. Most of xylanases are susceptible to be inactive when suffering high temperature and high alkaline process. Therefore, it is necessary to develop a high amount of effective thermoalkaliphilic xylanases. This study aims to enhance thermoalkaliphilic xylanase production in Pichia pastoris through fermentation parameters optimization and novel efficient fed-batch strategy in high cell-density fermentation...
June 21, 2017: BMC Biotechnology
https://www.readbyqxmd.com/read/28627778/engineering-cho-cells-with-an-oncogenic-kit-improves-cells-growth-resilience-to-stress-and-productivity
#3
Mohamed Mahameed, Boaz Tirosh
An optimized biomanufacturing process in mammalian cells is contingent on the ability of the producing cells to reach high viable cell densities. In addition, at the peak of growth, cells need to continue producing the biological entity at a consistent quality. Thus, engineering cells with robust growth performance and resilience to variable stress conditions is highly desirable. The tyrosine kinase receptor, KIT, plays a key role in cell differentiation and the survival of several immune cell types. Its oncogenic mutant, D816V, endows cells with high proliferation capacity and resistance to kinase inhibitors...
June 19, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28619018/efficient-soluble-expression-of-disulfide-bonded-proteins-in-the-cytoplasm-of-escherichia-coli-in-fed-batch-fermentations-on-chemically-defined-minimal-media
#4
Anna Gąciarz, Narendar Kumar Khatri, M Lourdes Velez-Suberbie, Mirva J Saaranen, Yuko Uchida, Eli Keshavarz-Moore, Lloyd W Ruddock
BACKGROUND: The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase...
June 15, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28606738/engineering-high-level-production-of-fatty-alcohols-by-saccharomyces-cerevisiae-from-lignocellulosic-feedstocks
#5
Leo d'Espaux, Amit Ghosh, Weerawat Runguphan, Maren Wehrs, Feng Xu, Oliver Konzock, Ishaan Dev, Melissa Nhan, Jennifer Gin, Amanda Reider Apel, Christopher J Petzold, Seema Singh, Blake A Simmons, Aindrila Mukhopadhyay, Héctor García Martín, Jay D Keasling
Fatty alcohols in the C12-C18 range are used in personal care products, lubricants, and potentially biofuels. These compounds can be produced from the fatty acid pathway by a fatty acid reductase (FAR), yet yields from the preferred industrial host Saccharomyces cerevisiae remain under 2% of the theoretical maximum from glucose. Here we improved titer and yield of fatty alcohols using an approach involving quantitative analysis of protein levels and metabolic flux, engineering enzyme level and localization, pull-push-block engineering of carbon flux, and cofactor balancing...
June 10, 2017: Metabolic Engineering
https://www.readbyqxmd.com/read/28606596/mini-review-recombinant-production-of-tailored-bio-pharmaceuticals-in-different-bacillus-strains-and-future-perspectives
#6
REVIEW
Antonia Lakowitz, Thibault Godard, Rebekka Biedendieck, Rainer Krull
Bio-pharmaceuticals like antibodies, hormones and growth factors represent about one-fifth of commercial pharmaceuticals. Host candidates of growing interest for recombinant production of these proteins are strains of the genus Bacillus, long being established for biotechnological production of homologous and heterologous proteins. Bacillus strains benefit from development of efficient expression systems in the last decades and emerge as major industrial workhorses for recombinant proteins due to easy cultivation, non-pathogenicity and their ability to secrete recombinant proteins directly into extracellular medium allowing cost-effective downstream processing...
June 9, 2017: European Journal of Pharmaceutics and Biopharmaceutics
https://www.readbyqxmd.com/read/28603019/friability-testing-as-a-new-stress-stability-assay-for-biopharmaceuticals
#7
Tetsuo Torisu, Takahiro Maruno, Saki Yoneda, Yoshinori Hamaji, Shinya Honda, Tadayasu Ohkubo, Susumu Uchiyama
A cycle of dropping and shaking a vial containing antibody solution was reported to induce aggregation. In this study, antibody solutions in glass prefillable syringes with or without silicone oil lubrication were subjected to the combined stresses of dropping and shaking, using a friability testing apparatus. Larger numbers of subvisible particles were generated, regardless of silicone oil lubrication, upon combination stress exposure than that with shaking stress alone. Nucleation of antibody molecules upon perturbation by an impact of the dropping, and/or adsorption of antibody molecules to the syringe surface followed by film formation and antibody film desorption were considered key steps in the particle formation promoted by combination stress...
June 8, 2017: Journal of Pharmaceutical Sciences
https://www.readbyqxmd.com/read/28579488/effective-cell-free-drug-screening-protocol-for-protein-protein-interaction
#8
Shaked Ashkenazi, Alexander Plotnikov, Anat Bahat, Rivka Dikstein
Specific protein-protein interaction (PPI) is an essential feature of many cellular processes however, targeting these interactions by small molecules is highly challenging due to the nature of the interaction interface. Thus, screening for PPI inhibitors requires enormous number of compounds. Here we describe a simple and improved protocol designed for a search of direct PPI inhibitors. We engineered a bacterial expression system for the split-Renilla luciferase (RL) complementation assay that monitors PPI...
June 1, 2017: Analytical Biochemistry
https://www.readbyqxmd.com/read/28556626/media-development-for-large-scale-agrobacterium-tumefaciens-culture
#9
Ingrid K Leth, Karen A McDonald
A chemically defined media was developed for growing Agrobacterium tumefaciens at large scale for commercial production of recombinant proteins by transient expression in plants. Design of experiments was used to identify major and secondary effects of ten media components: sucrose, ammonium sulfate ((NH4 )2 SO4 ), magnesium sulfate heptahydrate (MgSO4 *7H2 O), calcium chloride dihydrate (CaCl2 *2H2 O), iron (II) sulfate heptahydrate (FeSO4 *7H2 O), manganese (II) sulfate monohydrate (MnSO4 *H2 O), zinc sulfate heptahydrate (ZnSO4 *7H2 O), sodium chloride (NaCl), potassium chloride (KCl) and a sodium/potassium phosphate buffer (Na2 HPO4 /KH2 PO4 )...
May 27, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/28551876/identification-and-characterization-of-a-new-agar-degrading-strain-with-the-novel-properties-of-saccharides-inhibition-and-nitrogen-fixation
#10
Hao Wu, Guiguang Chen, Yaxi Bian, Wei Zeng, Bihong Sun, Zhiqun Liang
In this study, a new agar-degrading strain was isolated from soil with agar as a sole carbon source and energy. Based on its morphological, physiological, biochemical characterization and 16S rDNA sequence, the strain was identified as Streptomyces lavendulae UN-8. The extracellular agarase activity reached 0.03 U/ml after fermentation in shake flask (250 ml), which was close to other reported non-marine microorganisms. Furthermore, it is interesting that the growth of UN-8 would be inhibited by glucose (40 g/L) and maltose (40 g/L) with the inhibitory rate of 100% and 70%, respectively...
June 2017: Journal of Microbiology / the Microbiological Society of Korea
https://www.readbyqxmd.com/read/28547769/evaluation-of-piggybac-mediated-cho-pools-to-enable-material-generation-to-support-glp-toxicology-studies
#11
Yashas Rajendra, Sowmya Balasubramanian, Neil A McCracken, Dawn L Norris, Zhirui Lian, Matthew G Schmitt, Christopher C Frye, Gavin C Barnard
Generating purified protein for GLP toxicology studies (GLP-Tox) represents an important and often rate limiting step in the biopharmaceutical drug development process. Toxicity testing requires large amounts of therapeutic protein (>100g), typically produced in a single 500L - 2,500L bioreactor, using the final CHO clonally-derived cell line (CDCL). One approach currently used to save time is to manufacture GLP-Tox material using pools of high-producing CHO CDCLs instead of waiting for the final CDCL. Recently, we reported CHO pools producing mAb titers >7 g/L using piggyBac-mediated gene integration (PB CHO pools)...
May 25, 2017: Biotechnology Progress
https://www.readbyqxmd.com/read/28521794/in-vitro-synthesis-of-9-10-dihydroxyhexadecanoic-acid-using-recombinant-escherichia-coli
#12
Anees Kaprakkaden, Preeti Srivastava, Virendra Swarup Bisaria
BACKGROUND: Hydroxy fatty acids are widely used in food, chemical and cosmetic industries. A variety of dihydroxy fatty acids have been synthesized so far; however, no studies have been done on the synthesis of 9,10-dihydroxyhexadecanoic acid. In the present study recombinant E. coli has been used for the heterologous expression of fatty acid hydroxylating enzymes and the whole cell lysate of the induced culture was used for in vitro production of 9,10-dihydroxyhexadecanoic acid. RESULTS: A first of its kind proof of principle has been successfully demonstrated for the production of 9,10-dihydroxyhexadecanoic acid using three different enzymes viz...
May 18, 2017: Microbial Cell Factories
https://www.readbyqxmd.com/read/28482913/critical-attributes-of-human-early-mesenchymal-stromal-cell-laden-microcarrier-constructs-for-improved-chondrogenic-differentiation
#13
Youshan Melissa Lin, Jialing Lee, Jessica Fang Yan Lim, Mahesh Choolani, Jerry Kok Yen Chan, Shaul Reuveny, Steve Kah Weng Oh
BACKGROUND: Microcarrier cultures which are useful for producing large cell numbers can act as scaffolds to create stem cell-laden microcarrier constructs for cartilage tissue engineering. However, the critical attributes required to achieve efficient chondrogenic differentiation for such constructs are unknown. Therefore, this study aims to elucidate these parameters and determine whether cell attachment to microcarriers throughout differentiation improves chondrogenic outcomes across multiple microcarrier types...
May 8, 2017: Stem Cell Research & Therapy
https://www.readbyqxmd.com/read/28470602/high-yield-of-recombinant-protein-in-shaken-e-coli-cultures-with-enzymatic-glucose-release-medium-enpresso-b
#14
Kaisa Ukkonen, Antje Neubauer, Vinit J Pereira, Antti Vasala
Expression of recombinant proteins in sufficient quantities is essential for protein structure-function studies. The most commonly used method for recombinant protein production is overexpression in E. coli cultures. However, producing high yields of functional proteins in E. coli can be a challenge in conventional shaken cultures. This is often due to nonoptimal growth conditions, which result in low cell yields and insoluble or incorrectly folded target protein. To overcome the shortcomings of shake flask cultivation, we present a cultivation method based on enzymatic glucose delivery...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28465212/cell-culture-media-supplemented-with-raffinose-reproducibly-enhances-high-mannose-glycan-formation
#15
David Brühlmann, Anais Muhr, Rebecca Parker, Thomas Vuillemin, Blanka Bucsella, Franka Kalman, Serena Torre, Fabio La Neve, Antonio Lembo, Tobias Haas, Markus Sauer, Jonathan Souquet, Hervé Broly, Jürgen Hemberger, Martin Jordan
Glycosylation plays a pivotal role in pharmacokinetics and protein physiochemical characteristics. In particular, effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) can be desired, and it has been described that high-mannose species exhibited enhanced ADCC. In this work we present the trisaccharide raffinose as a novel cell culture medium supplement to promote high mannose N-glycans in fed-batch cultures, which is sought after in the development of biosimilars to match the quality profile of the reference medicinal product (RMP) also...
April 29, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28436005/fast-and-reliable-strain-characterization-of-streptomyces-lividans-through-micro-scale-cultivation
#16
Joachim Koepff, Matthias Keller, Konstantinos C Tsolis, Tobias Busche, Christian Rückert, Mohamed B Hamed, Jozef Anné, Joern Kalinowski, Wolfgang Wiechert, Anastassios Economou, Marco Oldiges
Filamentous organisms of the genus Streptomyces play an important role in industrial production processes, due to their extensive secondary metabolism variability, as well as their ability to secrete efficiently large amounts of (heterologous) proteins. While genetic engineering tools are available to rapidly build up large strain libraries, the subsequent strain screening and bioprocess development still constitutes a bottleneck. This is due to the lack of reliable parallelized and accelerated cultivation techniques for morphologically challenging organisms...
April 24, 2017: Biotechnology and Bioengineering
https://www.readbyqxmd.com/read/28420336/reduced-changes-in-protein-compared-to-mrna-levels-across-non-proliferating-tissues
#17
Kobi Perl, Kathy Ushakov, Yair Pozniak, Ofer Yizhar-Barnea, Yoni Bhonker, Shaked Shivatzki, Tamar Geiger, Karen B Avraham, Ron Shamir
BACKGROUND: The quantitative relations between RNA and protein are fundamental to biology and are still not fully understood. Across taxa, it was demonstrated that the protein-to-mRNA ratio in steady state varies in a direction that lessens the change in protein levels as a result of changes in the transcript abundance. Evidence for this behavior in tissues is sparse. We tested this phenomenon in new data that we produced for the mouse auditory system, and in previously published tissue datasets...
April 18, 2017: BMC Genomics
https://www.readbyqxmd.com/read/28410897/efficient-production-of-antibody-fab-fragment-by-transient-gene-expression-in-insect-cells
#18
Keita Mori, Hirotsugu Hamada, Takafumi Ogawa, Yuki Ohmuro-Matsuyama, Tomohisa Katsuda, Hideki Yamaji
Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B...
April 11, 2017: Journal of Bioscience and Bioengineering
https://www.readbyqxmd.com/read/28402216/a-novel-and-efficient-immunoglobulin-y-extraction-method-using-poloxamer-polyethylene-glycol
#19
Cui Li, Hao Ren, Rüdiger Schade, Xiaoying Zhang
Although many IgY extraction methods (such as polyethylene glycol (PEG) precipitation method, octanoic acid method, water dilution method, etc.) have been established, there is still industrial drive and real need in developing scale-up IgY production methods. Some previous studies have reported that poloxamer degreasing method shows very good result in IgY extraction from egg yolk with high degreasing speed, harmlessness, simpleness in operation and minimal effect on antibody titer. In this study, we developed a new method, poloxamer-PEG method, to obtain functional IgY with high purity and yield...
April 12, 2017: Preparative Biochemistry & Biotechnology
https://www.readbyqxmd.com/read/28389137/sugar-perceived-healthfulness-and-satiety-when-does-a-sugary-preload-lead-people-to-eat-more
#20
Naomi Mandel, Daniel Brannon
In this research, we examine the interplay between physiological and psychological factors that determine whether the sugar level of a preload increases or decreases consumption on a subsequent snack-eating task. In study 1, participants who drank a high-sugar protein shake (which they believed to be healthy) consumed more subsequent snacks than participants who drank a low-sugar protein shake. Study 2 replicated these findings, but only when the shake was labeled as "healthy." When the shake was labeled as "indulgent," the effect was mitigated...
April 4, 2017: Appetite
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