Skip to main content

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

  • 1057 Accesses

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


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)

This is a preview of subscription content, access via your institution.

Buying options

USD   49.95
Price excludes VAT (USA)
  • DOI: 10.1007/978-1-0716-0775-6_8
  • Chapter length: 11 pages
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
USD   149.00
Price excludes VAT (USA)
  • ISBN: 978-1-0716-0775-6
  • Instant PDF download
  • Readable on all devices
  • Own it forever
  • Exclusive offer for individuals only
  • Tax calculation will be finalised during checkout
Softcover Book
USD   199.99
Price excludes VAT (USA)
Hardcover Book
USD   279.99
Price excludes VAT (USA)
Fig. 1

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more


  1. Albertsson PA (1986) Partition of cell particles and macromolecules. Wiley, New York, NY

    Google Scholar 

  2. Diamond AD, Hsu JT (1992) Aqueous two-phase systems for biomolecule separation. Adv Biochem Eng Biotechnol 47:89–135

    CAS  PubMed  Google Scholar 

  3. Haraguchi LH, Mohamed RS, Loh W et al (2004) Phase equilibrium and insulin partitioning in aqueous two-phase systems containing block copolymers and potassium phosphate. Fluid Phase Equilibr 215:1–15

    CrossRef  CAS  Google Scholar 

  4. Reh G, Nerli B, Pico G (2002) Isolation of alpha-1 antitrypsin from human plasma by partitioning in aqueous biphasic systems of polyethyleneglycol-phosphate. J Chromatogr B 780:389–396

    CrossRef  CAS  Google Scholar 

  5. Cavalcanti MTH, Porto TS, Neto BB et al (2006) Aqueous two-phase systems extraction of α-toxin from Clostridium perfigens type A. J Chromatogr B 833:135–140

    CrossRef  CAS  Google Scholar 

  6. Rosa PAJ, Azevedo AM, Ferreira IF et al (2007) Affinity partitioning of human antibodies in aqueous two-phase systems. J Chromatogr A 1162:103–113

    CrossRef  CAS  PubMed  Google Scholar 

  7. Chethana S, Nayak CA, Raghavarao KSMS (2007) Aqueous two-phase extraction for purification and concentration of betalains. J Food Eng 81:679–687

    CrossRef  CAS  Google Scholar 

  8. Patil G, Raghavarao KSMS (2007) Aqueous two-phase extraction for purification of C-phycocyanin. Biochem Eng J 34:156–164

    CrossRef  CAS  Google Scholar 

  9. Johansson H, Ishii M, Minaguti M et al (2008) Separation and partitioning of green fluorescent protein from Escherichia coli homogenate in poly(ethylene glycol)/sodium-poly(acrylate) aqueous two-phase systems. Sep Purif Technol 62:166–174

    CrossRef  CAS  Google Scholar 

  10. Soares RR, Azevedo AM, Van Alstine JM, Aires-Barros MR (2015) Partitioning in aqueous two-phase systems: analysis of strengths, weaknesses, opportunities and threats. Biotechnol J 10:1158–1169

    CrossRef  CAS  PubMed  Google Scholar 

  11. Phong WN, Le CF, Show PL, Chang JS, Ling TC (2017) Extractive disruption process integration using ultrasonication and an aqueous two-phase system for protein recovery from Chlorella sorokiniana. Eng Life Sci 17:357–369

    CrossRef  CAS  PubMed  Google Scholar 

  12. Iqbal M et al (2016) Aqueous two-phase system (ATPS): an overview and advances in its applications. Biol Proced Online 18:18

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lee JW, Forciniti D (2010) Purification of human antibodies from transgenic corn using aqueous two-phase systems. Biotechnol Prog 26:159–167

    CrossRef  CAS  PubMed  Google Scholar 

  14. Singh N, Sibylle H (2017) Downstream processing technologies/capturing and final purification. In: Ghose TK, Fiechter A, Blakebrough N (eds) Advances in biochemical engineering. Springer, Berlin, pp 1–64

    Google Scholar 

  15. Tran R, Titchener-Hooker NJ, Lacki K (2011) Aqueous two phase extraction augmented precipitation process for purification of therapeutic proteins Patent application number: 20110257378

    Google Scholar 

  16. Bhambure R, Sharma R, Gupta D et al (2013) A novel aqueous two phase assisted platform for efficient removal of process related impurities associated with E. coli based biotherapeutic protein products. J Chromatogr A 1307:49–57

    CrossRef  CAS  PubMed  Google Scholar 

  17. Rathore A (2012) A process for purification of Recombinant granulocyte colony stimulating factor. Patent application number: 1880/DEL 2012

    Google Scholar 

  18. GuanY LTH, Garcia-Lisbona MN et al (1995) New approaches to aqueous polymer systems: theory, thermodynamics and applications to biomolecular separations. Pure Appl Chem 67:955–962

    CrossRef  Google Scholar 

  19. Mokhtarani B, Karimzadeh R, Amini MH et al (2008) Partitioning of Ciprofloxacin in aqueous two-phase system of poly(ethylene glycol) and sodium sulphate. Biochem Eng J 38:241–247

    CrossRef  CAS  Google Scholar 

  20. Zaslavsky BS (1995) Aqueous two-phase partitioning: physical chemistry and bioanalytical applications. Marcel Dekker Inc., New York, NY

    Google Scholar 

  21. Kaul A (2000) Phase diagram. In: Kaul A (ed) Methods in biotechnology, vol. 11: aqueous two-phase systems: methods and protocols. Humana, Totowa, NJ

    Google Scholar 

  22. Ferreira GB, Evangelista AF, Junio JBS et al (2007) Partitioning optimization of proteins from Zea mays malt in ATPS PEG 6000/CaCl2. Braz Arch Biol Technol 50:557–564

    CrossRef  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Anurag S. Rathore .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Verify currency and authenticity via CrossMark

Cite this protocol

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.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0774-9

  • Online ISBN: 978-1-0716-0775-6

  • eBook Packages: Springer Protocols