Minimizing the Influence of Fluorescent Tags on IgG Partition in PEG-Salt Aqueous Two-Phase Systems for Rapid Screening Applications

Biotechnology Journal
Mariana N São PedroR R G Soares

Abstract

Aqueous two-phase extraction (ATPE) has been showing significant potential in the biopharmaceutical industry, allowing the selective separation of high-value proteins directly from unclarified cell culture supernatants. In this context, effective high-throughput screening tools are critical to perform a rapid empirical optimization of operating conditions. In particular, microfluidic ATPE screening devices, coupled with fluorescence microscopy to continuously monitor the partition of fluorophore-labeled proteins, have been recently demonstrated to provide short diffusion distances and rapid partition, using minimal reagent volumes. Nevertheless, the currently overlooked influence of the labeling procedure on partition must be carefully evaluated to validate the extrapolation of results to the unlabeled molecule. Here, three fluorophores with different global charge and reactivity selected to label immunoglobulin G (IgG) at degrees of labeling (DoL) ranging from 0.5 to 7.6. Labeling with BODIPY FL maleimide (DoL = 0.5), combined with tris(2-carboxyethyl) phosphine (TCEP) to generate free thiol groups, is the most promising strategy to minimize the influence of the fluorophore on partition. In particular, the partition coefficien...Continue Reading

References

Jan 1, 1982·Journal of Immunological Methods·E SchauensteinF Dachs
Aug 12, 1998·Journal of Chromatography. B, Biomedical Sciences and Applications·H O JohanssonCharles A Haynes
Jun 15, 2007·Journal of Biotechnology·A M AzevedoM R Aires-Barros
Jul 10, 2007·Journal of Chromatography. a·Ana C A RoqueM Angela Taipa
Sep 22, 2009·The Analyst·Masatoshi TsukamotoEiichi Tamiya
Aug 19, 2010·Biotechnology and Bioengineering·Stefan A OelmeierJürgen Hubbuch
Sep 21, 2010·Biotechnology Progress·Lisong Nathan MaoJohn Pieracci
Sep 8, 2011·Lab on a Chip·Steffen Hardt, Thomas Hahn
Apr 13, 2012·Journal of Chromatography. a·J A Asenjo, B A Andrews
Jun 30, 2012·Journal of Chromatography. a·D F C SilvaM R Aires-Barros
Aug 14, 2012·Journal of Separation Science·Matthias WiendahlJürgen Hubbuch
Feb 1, 2015·Journal of Chromatography. a·Christopher Ladd EffioJürgen Hubbuch
Dec 15, 2016·Methods : a Companion to Methods in Enzymology·Inês F PintoV Chu

Citations

Jan 11, 2020·Frontiers in Bioengineering and Biotechnology·Nagesh K Tripathi, Ambuj Shrivastava
Jun 25, 2020·Biotechnology Journal·Raquel Dos SantosAna C A Roque
Sep 18, 2020·Advances in Colloid and Interface Science·Shixiang LiuHailin Cong

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