Automated high-throughput dense matrix protein folding screen using a liquid handling robot combined with microfluidic capillary electrophoresis

An, Philip; Winters, Dwight; Walker, Kenneth W.

doi:10.1016/j.pep.2015.11.015

Cited by 5 publications

(3 citation statements)

References 24 publications

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“…of the assay calibration standard, still impact the final results. Systematic differences between the pipetting techniques of operators were reported to cause significant deviations in results [1]. A dilution-dependent positive bias at low dilution factors (up to 1:10) and a negative bias of samples diluted more than 1:100 was reported [13] and also observed in our data (Fig.…”

Section: Discussionsupporting

confidence: 84%

“…Since the beginning of the 1990s, especially after the turn of the millennium, automated liquid handling systems enabled high-throughput methods and thus revolutionized laboratory work. This is highlighted by numbers of publications in different fields of application such as nucleic acid synthesis and analysis, protein refolding, production host clone screening, process development, diagnostics, cell culture and others [1][2][3][4][5][6][7][8][9]. However, setting up fully automated methods is laborious, time-consuming [10,11] and investment costs for fully automated equipment are high.…”

Section: Introductionmentioning

confidence: 99%

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Semi-automation of process analytics reduces operator effect

Christler

Felföldi

Mosor

et al. 2019

Bioprocess Biosyst Eng

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The aim of this study was to semi-automate process analytics for the quantification of common impurities in downstream processing such as host cell DNA, host cell proteins and endotoxins using a commercial liquid handling station. By semiautomation, the work load to fully analyze the elution peak of a purification run was reduced by at least 2.41 h. The relative standard deviation of results among different operators over a time span of up to 6 months was at the best reduced by half, e.g. from 13.7 to 7.1% in dsDNA analysis. Automation did not improve the reproducibility of results produced by one operator but released time for data evaluation and interpretation or planning of experiments. Overall, semi-automation of process analytics reduced operator-specific influence on test results. Such robust and reproducible analytics is fundamental to establish process analytical technology and get downstream processing ready for Quality by Design approaches.

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Section: Discussionsupporting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Semi-automation of process analytics reduces operator effect

Christler

Felföldi

Mosor

et al. 2019

Bioprocess Biosyst Eng

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show abstract

“…Consequently, several refolding methods have been extensively reported (e.g. dilution, dialysis, chromatography and microfluidic chips 10 – 14 ). However, as many proteins can only be refolded under very specific conditions, the development of systematic screening methods that can screen multiple refolding conditions in parallel is still challenging.…”

Section: Introductionmentioning

confidence: 99%

A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables

Wang

Oosterwijk

Ali

et al. 2017

Sci Rep

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Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which “helper” molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the “helper” molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.

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A High-Throughput Automated Protein Folding System

Walker

Winters

2019

Methods in Molecular Biology

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Automated high-throughput dense matrix protein folding screen using a liquid handling robot combined with microfluidic capillary electrophoresis

Cited by 5 publications

References 24 publications

Semi-automation of process analytics reduces operator effect

Semi-automation of process analytics reduces operator effect

A Systematic Protein Refolding Screen Method using the DGR Approach Reveals that Time and Secondary TSA are Essential Variables

A High-Throughput Automated Protein Folding System

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