Review Extraction and Purification of Bioproducts and Nanoparticles using Aqueous Two-Phase Systems Strategies Aqueous Two-Phase Systems (ATPS) is a primary recovery technique that has shown great potential for the efficient extraction and purification of high value biological compounds. The main advantages of this technique include scaling up feasibility, process integration capability and biocompatibility. In this review, the efficient use of ATPS for the extraction of proteins, genetic material, low molecu- lar weight compounds, bioparticles, nanoparticles and cells is highlighted. The important role of ATPS in process integration, i.e., extractive conversion, extrac- tive fermentation, cell disruption integrated with product recovery, and extractive purification, is discussed. A novel approach to protein molecular characterization combining ATPS and 2-dimension electrophoresis (2-DE) is introduced as a first step in the process development. Novel approaches for downstream processing using ATPS and dielectrophoresis are presented. Finally, trends concerning the application of ATPS strategies to address the future challenges of bioseparation are discussed. Keywords: Aqueous Two-Phase Systems, Biological Products, Extraction Received: February 2, 2008; accepted: March 24, 2008 DOI: 10.1002/ceat.200800068 1 Introduction Aqueous Two-Phase Systems (ATPS) is a liquid-liquid extrac- tion technique that has been used to establish bioprocesses for the primary recovery and partial purification of a variety of biological products, including proteins, genetic material, nanoparticles, low molecular weight products, cells and cell or- ganelles [1, 2]. The main advantages of this technique include scaling up feasibility, process integration capability and bio- compatibility. ATPS form when hydrophilic compounds such as some types of polymers (polyethylene glycol, dextran, poly- propylene glycol, etc.) and salts (phosphates, sulfates, citrates, etc.) are combined over certain critical concentrations, result- ing in the formation of two hydrophilic phases, Fig. 1. There are three main types of ATPS: (1) polymer-salt, (2) polymer- polymer, and (3) ATPS constructed with alternative com- pounds, e.g., ethylene oxide and propylene oxide copolymers (EOPO), hydroxylpropyl starch (HPS), iminoadiacetic acid (IAA), etc. [3]. Of the polymer-salt systems uses, polyethylene glycol (PEG)-potassium phosphate, is particularly preferred due to important advantages, including extensive characteriza- tion, low cost, and a wide range of applications [3]. The first studies involving ATPS date from the late 1950’s and early 1960’s, when Albertsson [4] demonstrated the great potential of this technique for the primary recovery of biological com- pounds. Furthermore, process integration and intensification can be achieved using strategies based upon ATPS resulting in optimized processes that are easy to scale up [1–3]. Process in- tegration results when one single unit operation can achieve the same process objective of two or more discrete processing stages. Consequently, a reduction of the total number of unit operations is possible. On the other hand, process intensifica- tion involves the development of strategies that result in pro- cess modification to maximize the flow of the biological sus- pensions that enter the process. Such strategies do not require an increase in either the total number of stages or size of the equipment. Bioprocess development using ATPS is limited by the poor understanding and characterization of the effect of the system parameters upon the partitioning of a particular compound. Commonly, the partition behavior of the target products and contaminants under different system parameters, e.g., tie-line length (TLL), phase volume ratio, V R , pH, and sample loading, etc., is experimentally evaluated as a first step in process devel- © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.cet-journal.com Jorge Benavides 1 Oscar Aguilar 1 Blanca H. Lapizco-Encinas 1 Marco Rito-Palomares 1 1 Departamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnología, Tecnológico de Monterrey, Mexico. – Correspondence: Prof. M. Rito-Palomares (mrito@itesm.mx), Depar- tamento de Biotecnología e Ingeniería de Alimentos, Centro de Biotecnología, Tecnológico de Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, 64849, Mexico. 838 Chem. Eng. Technol. 2008, 31, No. 6, 838–845