Review
Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing

https://doi.org/10.1016/j.tibtech.2009.01.004Get rights and content

The therapeutic use of proteins has created an increasing demand for feasible and economical methods for both up- and downstream processes. However, whereas upstream processes have attracted substantial investment and commercial attention, downstream processing has been overlooked, causing a production bottleneck that is shifting the costs of production. This review focuses on the use of aqueous two-phase extraction as an option for the downstream processing of therapeutic proteins. It is a potential and promising liquid–liquid extraction technique for the purification of biomolecules, such as monoclonal antibodies, growth factors and hormones, that combines a high selectivity and biocompatibility with an easy scale-up and continuous operation mode.

Section snippets

Current issues in the downstream processing of biopharmaceuticals

The demand for large quantities of therapeutic proteins (Box 1) for the treatment of diseases requiring high doses and/or chronic administration (e.g. 375 mg/m2/week of Rituximab for the treatment of mantel cell lymphoma [1]) has led to concerns about a potential shortfall in the manufacturing capacity, which, in turn, has intensified the pressure to improve cell culture productivity [2].

Advances in molecular biology have led to an increase in cell line productivity by optimizing media

Anything but chromatography?

Downstream processing usually encompasses four stages, namely, recovery, isolation, purification and polishing. However, it is relatively easy to identify the bottlenecks of the process. With recovery, isolation and polishing making up only ∼20% of the total downstream costs, the major process limitations are found in the selective purification steps, currently dominated by chromatography 5, 6, which accounts for >70% of the downstream costs, mainly owing to media cost and relatively long cycle

Aqueous two-phase extraction

Aqueous two-phase systems (ATPSs) are formed spontaneously upon mixing two aqueous solutions of structurally different components, such as two polymers or a polymer and a salt, above a certain critical concentration. Since the pioneering work of Albertsson [23], aqueous two-phase extraction has been successfully applied in the downstream processing of several biological compounds, such as proteins, nucleic acids and amino acids. Because the bulk of both phases consists mainly of water, and most

Monoclonal antibodies (mAbs)

mAbs have been described as nature's biological warheads in that they are able to target and eliminate foreign and abnormal agents from the body [36]. Their specificity for an antigen has enormous clinical value and makes them therapeutic drugs with blockbuster potential. Indeed, mAbs have emerged as one of the most rapidly growing and lucrative class of therapeutics, with revenues expected to increase from around US$6.84 billion in 2004 to US$13.77 billion by 2011 in the USA alone [37].

Polymer–salt systems for downstream processing of mAbs

One of the first reports describing the use of a polymer–salt ATPS for the purification of mAbs dates back to 1992 to the work of Sulk et al. [43], who described a three-step method for the isolation of an immunoglobulin G1 (IgG1) mAb from a hybridoma cell culture supernatant. Purification was achieved using an aqueous two-phase extraction procedure, followed by a concentration step and thiophilic adsorption chromatography. Using this process, the overall yield in IgG1 was 71%, with 90%

Polymer–polymer systems for mAb purification

In an ATPS composed of two structurally different polymers, such as PEG and dextran, most proteins (including antibodies) partition preferentially to the more hydrophilic, dextran-rich phase. Hence, to enhance the affinity of antibodies towards the PEG-rich phase, the two terminal hydroxyl groups of PEG molecules have been modified with a wide range of ligand molecules. This strategy is not feasible in polymer–salt systems because of the high concentration of salt, which masks any affinity or

Insulin-like growth factor I

Insulin-like growth factor I (IGF-I) is a polypeptide hormone that is similar in molecular structure to insulin, which has an important role in childhood growth and continues to have anabolic effects in adults. IGF-I regulates islet β-cell growth, survival and metabolism and protects against type 1 diabetes [51]. The purification of IGF-I by aqueous two-phase extraction is often referred to in the literature as the industry's ATPS success case 52, 53, 54, and a patent for it has been issued by

Human growth hormone

Human growth hormone (hGH) is a single polypeptide chain of 191 amino acids with two disulphide bonds with a molecular weight of ∼22000 Da and a pI near 5.3. It has been used for the treatment of hypopituitary dwarfism and all other conditions resulting from low levels of hGH production [57].

Purification of hGH from E. coli inclusion bodies using a combination of an in situ solubilization step with an aqueous two-phase extraction step has been described by Hart and coworkers [53]. An ATPS

Insulin

Insulin is a polypeptide hormone produced by the β cells of the pancreatic islets of Langerhans and is composed of two different chains linked together by disulphide bonds, with a total molecular weight of 6000 Da [59]. It has an essential role in regulating blood glucose levels and has an important effect on the metabolism of proteins and lipids [60].

The partitioning of insulin in PEG–citrate ATPSs has been investigated at different pH values and with different PEG molecular weights [61].

Challenges and future trends

It is increasingly being recognized that a broader strategic approach is needed to improve the overall process performance and to alleviate capacity bottlenecks in the downstream purification of biopharmaceuticals. One of the major advantages of large-scale aqueous two-phase extraction is its easy scale-up together with the possibility of continuous operation using traditional liquid–liquid extractors, which would overcome some of the limitations that chromatographic separations are facing. In

Acknowledgements

A.M.A. acknowledges the initiative ‘Ciência 2007’ of the Portuguese ministry for science, technology and higher education (http://www.mctes.pt/). P.A.J.R. and I.F.F. acknowledge ‘Fundação para a Ciência e Tecnologia’ for the PhD fellowships BD/25040/2005 and BD/38941/2007, respectively. A.M.A. also acknowledges Joana Carvalho for all the journal papers requested.

References (71)

  • J. Huddleston

    The molecular basis of partitioning in aqueous two-phase systems

    Trends Biotechnol.

    (1991)
  • M. Bensch

    High throughput screening techniques in downstream processing: preparation, characterization and optimization of aqueous two-phase systems

    Chem. Eng. Sci.

    (2007)
  • A.M. Azevedo

    Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography

    J. Chromatogr. B

    (2009)
  • P.A.J. Rosa

    Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies

    J. Chromatogr. A

    (2007)
  • G.M. Zijlstra

    Design of aqueous two-phase systems supporting animal cell growth: a first step toward extractive bioconversions

    Enzyme Microb. Technol.

    (1996)
  • K. Naganagouda et al.

    Aqueous two-phase extraction (ATPE): an attractive and economically viable technology for downstream processing of Aspergillus oryzae α-galactosidase

    Process Biochem.

    (2008)
  • A.M. Azevedo

    Partitioning of human antibodies in polyethylene glycol – sodium citrate aqueous two-phase systems

    Sep. Purif. Technol.

    (2009)
  • D. Platis et al.

    Development of an aqueous two-phase partitioning system for fractionating therapeutic proteins from tobacco extract

    J. Chromatogr. A

    (2006)
  • I.F. Ferreira

    Purification of human immunoglobulin G by thermoseparating aqueous two-phase systems

    J. Chromatogr. A

    (2008)
  • B. Sulk

    Application of phase partitioning and thiophilic adsorption chromatography to the purification of monoclonal antibodies from cell culture fluid

    J. Immunol. Methods

    (1992)
  • A.M. Azevedo

    Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction and size-exclusion chromatography

    J. Chromatogr. A

    (2008)
  • G. Birkenmeier

    Immobilized metal ion affinity partitioning, a method combining metal–protein interaction and partitioning of proteins in aqueous two-phase systems

    J. Chromatogr. A

    (1991)
  • G.M. Zijlstra

    Extractive bioconversions in aqueous two-phase systems

    Curr. Opin. Biotechnol.

    (1998)
  • A.M. Azevedo

    Affinity-enhanced purification of human antibodies by aqueous two-phase extraction

    Sep. Purif. Technol.

    (2009)
  • M. Rito-Palomares

    Practical application of aqueous two-phase partition to process development for the recovery of biological products

    J. Chromatogr. B

    (2004)
  • J.G.L.F. Alves

    Partitioning of whey proteins, bovine serum albumin and porcine insulin in aqueous two-phase systems

    J. Chromatogr. B

    (2000)
  • L.H. Haraguchi

    Phase equilibrium and insulin partitioning in aqueous two-phase systems containing block copolymers and potassium phosphate

    Fluid Phase Equil

    (2004)
  • O. Aguilar

    Direct comparison between ion-exchange chromatography and aqueous two-phase processes for the partial purification of penicillin acylase produced by E. coli

    J. Chromatogr. B

    (2006)
  • J. Ruan et al.

    Mantel cell lymphoma: current concept in biology and treatment

    Cancer Treat. Res.

    (2006)
  • G. Hodge

    Media development for mammalian cell culture

    Biopharm. Int.

    (2005)
  • Frost & Sullivan, (2004) Strategic Analysis of Downstream Processing in Biopharmaceutical Production, Frost &...
  • A.C.A. Roque

    Antibodies and genetically engineered related molecules: production and purification

    Biotechnol. Prog.

    (2004)
  • J. Thömmes et al.

    Alternatives to chromatographic separations

    Biotechnol. Prog.

    (2007)
  • U. Gottschalk

    The renaissance of protein purification

    Biopharm. Int.

    (2006)
  • T.J. Hobley

    Advances in high-gradient magnetic fishing for downstream and bioprocessing [abstract]

    J. Biotechnol.

    (2005)
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