Journal of Chromatography B, 893–894 (2012) 182–186 Contents lists available at SciVerse ScienceDirect Journal of Chromatography B jo u r n al hom epage: www.elsevier.com/locate/chromb Short communication PEGylation, detection and chromatographic purification of site-specific PEGylated CD133-Biotin antibody in route to stem cell separation Mirna González-González, Karla Mayolo-Deloisa, Marco Rito-Palomares ∗ Centro de Biotecnología-FEMSA, Tecnológico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur, Monterrey, NL 64849, Mexico a r t i c l e i n f o Article history: Received 9 November 2011 Accepted 2 March 2012 Available online 7 March 2012 Keywords: CD133 Antibody PEGylation Stem/progenitor cells separation Streptavidin–biotin conjugation a b s t r a c t Recovery and purification of stem cells are determining steps in order to obtain the purity and viability required for transplantation. In this context, immunochemical techniques have been widely preferred due to their high selectivity. CD133, a glycoprotein expressed by stem cells, is a well-used marker for iso- lation of neural stem cells. Transplantation of neural stem cells into patients can promote neural growth and improve neuronal functions. In this study, a new method for site-specific PEGylation of CD133- Biotin antibody is performed through streptavidin–biotin conjugation. Purification was carried out by ion-exchange chromatography. The characterization of the single PEGylated CD133-Biotin antibody was confirmed using electrophoresis with silver staining and I 2 –BaCl 2 for PEG detection. Moreover, online PEG quantification directly after the chromatographic step was conducted (in each fraction) to detect exact elution times of PEG. In conclusion, the novel CD133-Biotin antibody PEGylation strategy conducted in this study could be used as a process step in route to neural stem cell recovery and purification via the modification of existing techniques such as aqueous two phase systems, PEGylated affinity columns or fluidized chromatography. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Stem cell transplantation has emerged as a novel therapeutic alternative for several incurable diseases. In this sense, the recov- ery and purification of stem cells are crucial steps in order to supply the number of cells required and in the degree of purity needed. In other words, the success of a transplant procedure is to a large extent dependent on the effectiveness of the purification and recov- ery strategies. A desirable recovery method for stem cells has to guarantee high purity and should be sensitive, rapid, quantitative, scalable, non-or minimally invasive to preserve viability and dif- ferentiation capacity of the purified cells [1–3]. Currently, there are a wide range of methodologies for stem cell isolation. Neverthe- less, there is not a golden standard method that accomplishes all requirements [3–5]. One of the most exploited techniques for stem cell isolation has been the immunochemical, owing the high specificity conferred by the surface marker (cluster of differentiation CD) employed as molecular tagging. For example, one of the most recently used CD for identification of stem cells is the novel CD133 (also known as Abbreviations: CD, cluster of differentiation; IEC, ion exchange chromatogra- phy; PBS, phosphate buffered saline; PEG, poly(ethylene glycol); SDS–PAGE, sodium dodecyl sulphate polyacrylamide; UV, ultraviolet. ∗ Corresponding author. Tel.: +52 81 8328 4132; fax: +52 81 8328 4136. E-mail address: mrito@itesm.mx (M. Rito-Palomares). prominin-1). CD133 is a five-transmembrane glycoprotein [6] that appears to be a reliable marker for the isolation of neural stem cells [7] and has the ability to promote neural growth [8]. Antibody PEGylation has been used in the past decades to improve therapy treatments due to the benefits that the cova- lent attachment of poly(ethylene glycol) (PEG) to the antibody confers. These advantages include: increase circulating half-lives of antibodies; reduce antigenicity, immunogenicity and toxicity; improve solubility and bioavailability; and enhance proteolytic resistance [9]. Nevertheless, this technique has not been exploited for the recovery and purification of stem cells. In this manner, the conjugation of PEG to a specific antibody (i.e. CD133-Biotin antibody) could confer valuable properties that could be exploited afterwards in downstream process. For example, the recovery and purification of stem cells for clinical applications could be achieved through modified aqueous two phase systems [10], PEGylated affin- ity columns or fluidized chromatography with PEGylated matrixes. In this sense, a site specific conjugation should be used to preserve the affinity of the interaction between the antibody and antigen. Streptavidin–biotin conjugation is an alternative for a site-specific reaction which can be conducted by employing derivatized PEGs with biotin and biotinylated antibodies (i.e. CD133-Biotin). In this way, streptavidin (with four binding sites for biotin) can bridge two biotinylated reagents and form a site-specific PEGylated antibody. This reaction is highly specific and results in only one product. In the present study, the site-specific PEGylation of CD133- Biotin antibody is performed through the streptavidin–biotin 1570-0232/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2012.03.002