A fully automated high-throughput plasmid purification workstation for the generation of mammalian cell expression-quality DNA.

Autor:	Cohen M; Department of Biologics Automation and High-Throughput Technologies, Eli Lilly and Company Biotechnology Center, San Diego, CA, USA., Randolph DE; Department of Biologics Automation and High-Throughput Technologies, Eli Lilly and Company Biotechnology Center, San Diego, CA, USA., Lozano ME; Department of Biologics Automation and High-Throughput Technologies, Eli Lilly and Company Biotechnology Center, San Diego, CA, USA., Anderson PW; Department of Biologics Automation and High-Throughput Technologies, Eli Lilly and Company Biotechnology Center, San Diego, CA, USA., Crissman J; Department of Biologics Automation and High-Throughput Technologies, Eli Lilly and Company Biotechnology Center, San Diego, CA, USA., Triana FJ; Department of Biologics Automation and High-Throughput Technologies, Eli Lilly and Company Biotechnology Center, San Diego, CA, USA., Cujec T; Department of Biologics Automation and High-Throughput Technologies, Eli Lilly and Company Biotechnology Center, San Diego, CA, USA. Electronic address: cujecth@lilly.com.
Jazyk:	angličtina
Zdroj:	SLAS technology [SLAS Technol] 2022 Aug; Vol. 27 (4), pp. 227-236. Date of Electronic Publication: 2022 Feb 06.
DOI:	10.1016/j.slast.2022.01.005
Abstrakt:	Early-stage antibody discovery and engineering typically require the cloning, expression, and screening of large numbers of proteins. Normally, DNA fragments encoding proteins of interest are cloned into extra-chromosomal plasmids that are amplified in Escherichia coli. Following purification from the bacteria, the plasmids are introduced into appropriate cells, and the expressed recombinant proteins screened for desired binding or function in a high-throughput manner. Even in a 96-well plate format, plasmid purification from E. coli is typically a labor intensive and time-consuming process. To further accelerate our existing biotherapeutic discovery workflows we designed, qualified, and enabled a fully integrated high-throughput plasmid purification and quantification workstation which we have termed AMPS (Automated Miniprep Plasmid Station). Using components from a commercially available kit, AMPS can purify plasmid preparations from twenty 96-deep-well plates of E. coli cultures, measure DNA absorbance at 260 nm, calculate plasmid concentrations, and prepare 96-deep-well plates for mammalian expression in an operator-independent manner. Plasmid yields and concentrations are equivalent to those obtained off-line. Furthermore, the quality of the DNA purified on the AMPS is equivalent to that obtained off-line in terms of DNA topology, and absence of contaminating bacterial chromosomal DNA and RNA. Most importantly, plasmids purified on the AMPS provide similar antibody titers following transfection in CHO cells as plasmids purified off-line. The AMPS bridges high-throughput E. coli colony picking capabilities typically available in an automation lab with downstream CHO expression needs and will facilitate screening of large numbers of biotherapeutics in binding and cell assay screens. (Copyright © 2022. Published by Elsevier Inc.)
Databáze:	MEDLINE
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