9 Perspective Received: 13 May 2016 Revised: 16 June 2016 Accepted article published: 21 June 2016 Published online in Wiley Online Library: 20 July 2016 (wileyonlinelibrary.com) DOI 10.1002/jctb.5040 Monolithic chromatography: insights and practical perspectives Mirna González-González, † José González-Valdez, † Karla Mayolo-Deloisa † and Marco Rito-Palomares * Abstract The use of monoliths, one-piece porous separation materials, as column packaging media is known as monolithic chromatogra- phy and this technology has been widely exploited to improve the separation profile of high-valued biomolecules from complex biological matrices. Monoliths possess a wide range of processing advantages over commonly used chromatographical resins, including: fast flow rates, short processing times, sufficient surface area interactions, and enhanced thoroughness. The appli- cation of three different types of monolithic supports (hydrophobic, ion exchange and affinity) as an alternative to overcome traditional drawbacks in bioseparations will be presented as a practical perspective for the recovery of PEGylated proteins, stem cells or industrial enzymes. In our experience, the use of monolithic matrixes in the separation of high-valued biological materi- als has greatly simplified the usual problems observed in traditional chromatographic separations, resulting in a highly efficient downstream separation procedure. © 2016 Society of Chemical Industry Keywords: monolithic chromatography; PEGylated proteins; stem cells; lacasse INTRODUCTION The growing need in biotechnological markets to obtain highly-purified biomolecules, especially proteins, using fast and efficient methods has promoted the development of new downstream processing strategies. For many years, the main oper- ation used to separate and purify many of these biomolecules has been liquid chromatography. In conventional chromatography with polymeric supports, mass transfer and adsorption kinetics are very important. The procurement of high mass transfer rates requires large pores with high connectivity, no constrictions, and fast adsorption kinetics; while obtaining high capacities relies on having large surface areas and high selectivities within the resin. 1 Since mass transfer in particle based resins occurs through diffusion, purification of large molecules is usually very time consuming. For this reason and knowing that one of the key components of any chromatographic technique is the column, many different stationary phases have been introduced in the past decades but to further improve them, the use of chromatographic monoliths was introduced around 1990. 2 Chromatographic monoliths consist of a single piece of highly open porous material that forms a highly interconnected network of channels. 1,2 Monoliths can be used for all kinds of operating modes, except size exclusion chromatography. This is because monoliths for large biomolecules rely on convection, 3 while size exclusion chromatography is based on the differences between individual molecules in their diffusivity into the pores. 1 With extremely large biomolecules, monoliths are an excellent alter- native to conventional materials, providing higher surface areas and productivity. 1 Probably the most recognized monolith feature is convective based transport which has an important impact on the separation of large molecules having low diffusivity like pro- teins, polynucleotides or viruses. 4 In this context, our group has had some practical experiences in the recovery of macromolecules using monoliths, especially with the purification of PEGylated pro- teins, stem cells and industrial enzymes. The aim of this article is to show how the use of monoliths is an efficient alternative when compared with traditional chromato- graphic supports using hydrophobic interaction, ion exchange and affinity chromatography. The practical perspective for the separation of PEGylated forms of Ribonuclease A (RNase A), Lysozyme and  -Lactoglobulin directly from PEGylation reactions will be presented. In addition, practical experiences in the purifi- cation of laccase and the use of affinity monoliths for stem cells and separation of other PEGylated proteins will be presented to show some practical examples and perspectives in the use of this innovative technology. MONOLITHS INSIGHTS Principle Monoliths are considered 4th-generation chromatographic mate- rials. They are a sponge-like chromatographic media that consist of a single continuous porous organic or inorganic stationary sep- aration block. This stationary phase supports high convective flow rates through the interconnected channels, which enable laminar ∗ Correspondence to: M Rito-Palomares, Centro de Biotecnología-FEMSA, Tec- nológico de Monterrey. Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, México. Email: mrito@itesm.mx † These authors contributed equally to this work. Centro de Biotecnología-FEMSA, Tecnológico de Monterrey. Campus Monter- rey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, México. J Chem Technol Biotechnol 2017; 92:9–13 www.soci.org © 2016 Society of Chemical Industry