Incorporation of automated buffer exchange empowers high-throughput protein and plasmid purification for downstream uses.

Autor:	Kates PA; Integrated Micro-Chromatography Systems, Inc., 110 Centrum Drive, Irmo, SC 29063, United States., Cook JN; Integrated Micro-Chromatography Systems, Inc., 110 Centrum Drive, Irmo, SC 29063, United States., Ghan R; Hamilton Company, Inc., Reno, NV, United States., Nguyen HJ; Integrated Micro-Chromatography Systems, Inc., 110 Centrum Drive, Irmo, SC 29063, United States., Sitasuwan P; Integrated Micro-Chromatography Systems, Inc., 110 Centrum Drive, Irmo, SC 29063, United States., Lee LA; Integrated Micro-Chromatography Systems, Inc., 110 Centrum Drive, Irmo, SC 29063, United States. Electronic address: lee@imcstips.com.
Jazyk:	angličtina
Zdroj:	SLAS technology [SLAS Technol] 2023 Aug; Vol. 28 (4), pp. 243-250. Date of Electronic Publication: 2023 Feb 01.
DOI:	10.1016/j.slast.2023.01.005
Abstrakt:	The continued acceleration of time-to-market product development and rising demand for biotherapeutics have hastened the need for higher throughput within the biopharmaceutical industry. Automated liquid handlers (ALH) are increasingly popular due to flexible programming that enables processing of multiple samples with an array of functions. This flexibility is useful in streamlining research that requires chromatographic procedures to achieve product purity for downstream analysis. However, purification of biologics often requires additional off-deck buffer exchange steps due to undesirable elution conditions such as high acid or high salt content. Expanding the capability of ALHs to perform purification in sequence with buffer exchange would, therefore, increase workflow efficiency by eliminating the need for manual intervention, thus expediting sample preparation. Here we demonstrate two different automated purifications using pipet-based dispersive solid-phase extraction (dSPE). The first is an affinity purification of His-tagged proteins from bacterial lysate. The second is an anion-exchange purification of plasmid DNA. Both methods are followed by buffer exchange performed by an ALH. Percent recoveries for the three purified recombinant proteins ranged from 51 ± 1.2 to 86 ± 10%. The yields were inversely correlated to starting sample load and protein molecular weight. Yields for plasmid purification ranged between 11.4 ± 0.8 and 13.7 ± 0.9 µg, with the largest plasmid providing the highest yield. Both programs were rapid, with protein purification taking <80 min and plasmid purification <60 min. Our results demonstrate that high-quality, ready-to-use biologics can be obtained rapidly from a crude sample after two separate chromatographic processes without manual intervention. Competing Interests: Declaration of Competing Interest The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: L. Andrew Lee reports a relationship with Integrated Micro-Chromatography Systems, Inc. that includes: board membership, employment, and equity or stocks. Patrick A. Kates reports a relationship with Integrated Micro-Chromatography Systems, Inc. that includes: employment. Jordan N. Cook reports a relationship with Integrated Micro-Chromatography Systems, Inc. that includes: employment. Ryan Ghan is employed by Hamilton Company, which manufactures and sells the MicroLab STAR liquid-handling platform. (Copyright © 2023 Integrated micro-chromatography systems, Inc. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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