Immunoaffinity aqueous two-phase systems to establish novel bioprocesses for the primary recovery of CD133+ stem cells

A short processing time and efficient scale-up stem cell isolation bioprocess is essential to exploit the potential of these cells for the treatment of multiple chronic diseases. Various methodologies have been used for stem cell recovery, however, most of them present economical and/or time-consuming drawbacks. In this work, the characterization and optimization of immunoaffinity aqueous two-phase systems, a liquid-liquid based separation technology enhanced with the PEGylation of the antibody, was conducted with the aim of increasing the specificity for the recovery of CD133+ stem cells from human umbilical cord blood samples. The methodology consisted in evaluating the partitioning of the different PEGylated antibodies (amine, carboxyl, thiol, succinimidyl ester, methoxy PEG and maleimide) in three previously studied aqueous two-phase systems (ATPS); PEG-dextran (DEX), Ucon-DEX and Ficoll-DEX. Subsequently, an optimization step was accomplished to manipulate the partition behavior of the CD133/2-pure antibody to the desired phase in the selected systems by varying (increasing and decreasing) two parameters closely related with the partitioning of molecules in aqueous two-phase systems; tie-line length (TLL) and volume ratio (VR). Afterwards, the partitioning behavior of the six different PEGylated antibodies in the optimized systems was tested. According to the results, the PEGylation of the CD133/2-biotin antibody induced a favorable change with respect to the non-PEGylated one when Ucon-DEX system was used, fractionating it to both phases. Likewise, the optimization of the systems showed to be effective to induce a change in the partition preference of the antibody. The best results were obtained when Ucon-DEX or PEG-DEX systems with TLL 15% w/w or 20% w/w with VR 3 were combined. Finally, PEGylated antibodies were added to the selected optimized systems. Even though a shift in the fractionation preference of the PEGylated CD133/2-biotin antibody was achieved in the optimized systems, it was not the adequate partition to justify the evaluation of this immunoaffinity ATPS with human umbilical cord samples. Both PEGylation and optimization showed to be effective to induce a change in the partition preference of the antibody, however, further studies are required to find the optimal system composition that will fractionate 100% of the antibody to the contaminants opposite phase, making this system an ideal candidate to be tested for the selectivity of CD133+ stem cells.