Trends in Biotechnology
ReviewChromatography-free recovery of biopharmaceuticals through aqueous two-phase processing
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)
Alternative bioseparation operations: life beyond packed-bed chromatography
Curr. Opin. Biotechnol.
(2004)Downstream processing of monoclonal antibodies – application of platform approaches
J. Chromatogr. B
(2007)Smart polymers: physical forms and bioengineering applications
Prog. Polym. Sci.
(2007)Affinity-based methodologies and ligands for antibody purification: advances and perspectives
J. Chromatogr. A
(2007)Optimisation of aqueous two-phase extraction of human antibodies
J. Biotechnol.
(2007)Affinity partitioning of human antibodies in aqueous two-phase systems
J. Chromatogr. A
(2007)- et al.
Membrane separations in biotechnology
Curr. Opin. Biotechnol.
(2001) Monoliths as stationary phases for separation of proteins and polynucleotides and enzymatic conversion
J. Chromatogr. B
(2001)Membrane adsorbers as purification tools for monoclonal antibody purification
J. Chromatogr. B
(2007)- et al.
Integrated bioprocesses
Curr. Opin. Microbiol.
(2005)
The molecular basis of partitioning in aqueous two-phase systems
Trends Biotechnol.
High throughput screening techniques in downstream processing: preparation, characterization and optimization of aqueous two-phase systems
Chem. Eng. Sci.
Downstream processing of human antibodies integrating an extraction capture step and cation exchange chromatography
J. Chromatogr. B
Application of central composite design to the optimisation of aqueous two-phase extraction of human antibodies
J. Chromatogr. A
Design of aqueous two-phase systems supporting animal cell growth: a first step toward extractive bioconversions
Enzyme Microb. Technol.
Aqueous two-phase extraction (ATPE): an attractive and economically viable technology for downstream processing of Aspergillus oryzae α-galactosidase
Process Biochem.
Partitioning of human antibodies in polyethylene glycol – sodium citrate aqueous two-phase systems
Sep. Purif. Technol.
Development of an aqueous two-phase partitioning system for fractionating therapeutic proteins from tobacco extract
J. Chromatogr. A
Purification of human immunoglobulin G by thermoseparating aqueous two-phase systems
J. Chromatogr. A
Application of phase partitioning and thiophilic adsorption chromatography to the purification of monoclonal antibodies from cell culture fluid
J. Immunol. Methods
Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction and size-exclusion chromatography
J. Chromatogr. A
Immobilized metal ion affinity partitioning, a method combining metal–protein interaction and partitioning of proteins in aqueous two-phase systems
J. Chromatogr. A
Extractive bioconversions in aqueous two-phase systems
Curr. Opin. Biotechnol.
Affinity-enhanced purification of human antibodies by aqueous two-phase extraction
Sep. Purif. Technol.
Practical application of aqueous two-phase partition to process development for the recovery of biological products
J. Chromatogr. B
Partitioning of whey proteins, bovine serum albumin and porcine insulin in aqueous two-phase systems
J. Chromatogr. B
Phase equilibrium and insulin partitioning in aqueous two-phase systems containing block copolymers and potassium phosphate
Fluid Phase Equil
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
Mantel cell lymphoma: current concept in biology and treatment
Cancer Treat. Res.
Media development for mammalian cell culture
Biopharm. Int.
Antibodies and genetically engineered related molecules: production and purification
Biotechnol. Prog.
Alternatives to chromatographic separations
Biotechnol. Prog.
The renaissance of protein purification
Biopharm. Int.
Advances in high-gradient magnetic fishing for downstream and bioprocessing [abstract]
J. Biotechnol.
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