Application of an aqueous two-phase systems high-throughput screening method to evaluate mAb HCP separation

2010 ◽  
Vol 108 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Stefan A. Oelmeier ◽  
Florian Dismer ◽  
Jürgen Hubbuch
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Two Phase ◽  
Aqueous Two Phase Systems ◽  
Phase Systems

High-throughput screening-based selection and scale-up of aqueous two-phase systems for pDNA purification

2012 ◽  
Vol 35 (22) ◽  
pp. 3197-3207 ◽  
Author(s):  
Matthias Wiendahl ◽  
Stefan A. Oelmeier ◽  
Florian Dismer ◽  
Jürgen Hubbuch
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Scale Up ◽  
Two Phase ◽  
Aqueous Two Phase Systems ◽  
Phase Systems

scholarly journals High-throughput screening of aqueous two-phase systems for polyphenol extraction from A. nodosum: a green chemistry approach

2021 ◽  
Author(s):  
Alex Olivares-Molina ◽  
Brenda Parker
Keyword(s):  
Natural Products ◽  
Green Chemistry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Scale Up ◽  
Model System ◽  
Two Phase ◽  
Aqueous Two Phase Systems ◽  
Scale Down ◽  
Phase Systems

Brown macroalgae are an attractive third-generation feedstock of natural products, in order to design green chemistry-compliant processes and reduce the use of organic solvents in bioactive product extraction, aqueous two-phase systems (ATPS) was applied. This research aimed to develop a high-throughput screening (HTS) to recover polyphenols from Ascophyllum nodosum using ATPS. In total, 384 different 2-phase systems were assessed using an automated liquid-handling system to evaluate polyphenol recovery using a model system of phloroglucinol to establish an optimal 2-phase system for polyphenol partitioning. Various ratios of PEG:potassium phosphate solutions were explored to evaluate partitioning of polyphenols via a scale-down approach. Scale-down selected system showed a recovery of phloroglucinol of 62.9±12.0%, this system was used for scale-up trials. Scale-up studies confirmed that the HTS method was able to recover polyphenols with a 54.8±14.2% in the phloroglucinol model system. When the optimised ATPS system was tested with a polyphenol extract, 93.62±8.24% recovery was observed. When ATPS was applied to a fucoidan and alginate biorefinery residue, 88.40±4.59% polyphenol was recovered. These findings confirm that ATPS is a valuable addition to the bioprocess toolkit for sustainable extraction of natural products from macroalgae in a multiproduct biorefinery approach.

High-Throughput 3D Neural Cell Culture Analysis Facilitated by Aqueous Two-Phase Systems

MRS Advances ◽  
2017 ◽  
Vol 2 (45) ◽  
pp. 2435-2441 ◽  
Author(s):  
Kristin Robin Ko ◽  
Rishima Agarwal ◽  
John Frampton
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
High Throughput ◽  
Hydroxypropyl Methylcellulose ◽  
3D Culture ◽  
Neural Cell ◽  
Epifluorescence Microscopy ◽  
Two Phase ◽  
Aqueous Two Phase Systems ◽  
Phase Systems

ABSTRACTThe three-dimensional (3D) culture of neural cells in extracellular matrix (ECM) gels holds promise for modeling neurodegenerative diseases and pre-clinical evaluation of novel therapeutics. However, most current strategies for fabricating 3D neural cell cultures are not well suited to automated production and analysis. Here, we present a facile, replicable, 3D cell culture system that is compatible with standard laboratory equipment and high-throughput workflows. This system uses aqueous two-phase systems (ATPSs) to confine small volumes (5 and 10 μl) of a commonly used ECM hydrogel (Matrigel) into thin, discrete layers, enabling highly-uniform production of 3D neural cell cultures in a 96-well plate format. These 3D neural cell cultures can be readily analyzed by epifluorescence microscopy and microplate reader. Our preliminary results show that many common polymers used in ATPSs interfere with Matrigel gelation and instead form fibrous precipitates. However, 0.5% hydroxypropyl methylcellulose (HPMC) and 2.5% dextran 10 kDa (D10) were observed to retain Matrigel integrity and had minimal impact on cell viability. This novel system offers a promising yet accessible platform for high-throughput fabrication of 3D neural tissues using readily available and cost-effective materials.

High-throughput downstream process development for cell-based products using aqueous two-phase systems

2016 ◽  
Vol 1464 ◽  
pp. 1-11 ◽  
Author(s):  
Sarah Zimmermann ◽  
Sarah Gretzinger ◽  
Marie-Luise Schwab ◽  
Christian Scheeder ◽  
Philipp K. Zimmermann ◽  
...  
Keyword(s):  
High Throughput ◽  
Process Development ◽  
Downstream Process ◽  
Two Phase ◽  
Aqueous Two Phase Systems ◽  
Phase Systems

High-throughput downstream process development for cell-based products using aqueous two-phase systems (ATPS) - A case study

2016 ◽  
Vol 12 (2) ◽  
pp. 1600587 ◽  
Author(s):  
Sarah Zimmermann ◽  
Christian Scheeder ◽  
Philipp K Zimmermann ◽  
Are Bogsnes ◽  
Mattias Hansson ◽  
...  
Keyword(s):  
High Throughput ◽  
Process Development ◽  
Downstream Process ◽  
Two Phase ◽  
Aqueous Two Phase Systems ◽  
Phase Systems

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

Continuous purification of antibodies from cell culture supernatant with aqueous two-phase systems: From concept to process

2013 ◽  
Vol 8 (3) ◽  
pp. 352-362 ◽  
Author(s):  
Paula A. J. Rosa ◽  
Ana M. Azevedo ◽  
S. Sommerfeld ◽  
Martina Mutter ◽  
Werner Bäcker ◽  
...  
Keyword(s):  
Cell Culture ◽  
Culture Supernatant ◽  
Cell Culture Supernatant ◽  
Two Phase ◽  
Aqueous Two Phase Systems ◽  
Phase Systems
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