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Aqueous Two-Phase-Assisted Precipitation of Proteins: A Platform for Isolation of Process-Related Impurities from Therapeutic Proteins

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Part of the Methods in Molecular Biology book series (MIMB,volume 2178)

Abstract

Aqueous two-phase systems (ATPS) have been widely and successfully used in the purification of various biological macromolecules such as proteins, nucleic acids, antibiotics, and cell components. Interfacial precipitation of the product often results in lower recovery and selectivity of ATPS. Efficient resolubilization of the interfacial precipitate offers a way to improve the recovery as well as selectivity of ATPS systems.

In this protocol, we describe a method for aqueous two-phase-assisted precipitation and resolubilization of the recombinant human Granulocyte Colony Stimulating Factor (GCSF) for its selective isolation from E. coli host cell proteins as well as nucleic acids. This platform purification can be applied to other cytokines as well as most of the hydrophobic proteins that partition into the hydrophobic PEG-rich top phase. Recoveries of up to 100% of the product along with reduction of levels of E. coli host cell proteins (from 250–500 to 10–15 ppm) and of nucleic acids (from 15–20 to 5–15 ng/mL) were observed.

Key words

  • Aqueous two-phase systems (ATPS)
  • Interfacial precipitation
  • Host cell proteins (HCPs)
  • Deoxyribonucleic acid (DNA)
  • Granulocyte colony stimulating factor (GCSF)

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  • DOI: 10.1007/978-1-0716-0775-6_8
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Correspondence to Anurag S. Rathore .

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Rathore, A.S., Bhambure, R. (2021). Aqueous Two-Phase-Assisted Precipitation of Proteins: A Platform for Isolation of Process-Related Impurities from Therapeutic Proteins. In: Labrou, N.E. (eds) Protein Downstream Processing. Methods in Molecular Biology, vol 2178. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0775-6_8

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  • DOI: https://doi.org/10.1007/978-1-0716-0775-6_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0774-9

  • Online ISBN: 978-1-0716-0775-6

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