Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins

Abstract

Background: Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulinlike growth factor derivative.

Optimization of cell line development in the GS-CHO expression system using a high-throughput, single cell-based clone selection system

“…Optimization of the semi-solid medium formulation for single cell-based clone selection There are several reports of single cell-based clone selection using semi-solid media (27,31,32). However, they do not describe the medium formulation for single cell-based selection.…”

Utilization of non‐AUG initiation codons in a flow cytometric method for efficient selection of recombinant cell lines

“…Although there are several other screening strategies and technologies (Browne and Al-Rubeai, 2007;Caron et al, 2009;Serpieri et al, 2010) specifically designed to overcome the challenge of finding rare high expressing cells in the background of low and non-expressing cells, the versatility of the alternate start reporter strategy gives it several advantages over these systems. For instance, the pivotal role of flow cytometry in this method provides a considerable throughput advantage relative to the immunological detection of secreted product in semi solid media.…”

“…To address the need for greater yields, several different strategies have been pursued. These include utilizing stronger promoters in expression vectors (Gaillet et al, 2010;Prentice et al, 2007;Running Deer and Allison, 2004;Xia et al, 2006), better post-transfection selection systems (Sautter and Enenkel, 2005;van Blokland et al, 2007), and more efficient strategies for selecting clones with high expression levels (Bailey et al, 2002;Bohm et al, 2005;Brezinsky et al, 2003;Browne and Al-Rubeai, 2007;Caron et al, 2009). With respect to clone selection strategies, we and others have shown that IRES mediated expression of a reporter in conjunction with flow cytometry or magnetic separation methods can effectively isolate high expressing clones without requiring an antibody specific for the recombinant protein of interest (DeMaria et al, 2007;Gaines and Wojchowski, 1999;Liu et al, 2000;Sleiman et al, 2008).…”

Rapid automated selection of mammalian cell line secreting high level of humanized monoclonal antibody using Clone Pix FL system and the correlation between exterior median intensity and antibody productivity

“…Therefore, semi-solid media has been used to immobilize clones and a high throughput, automated colony picker (Clone Pix FL), use to efficiently isolate monoclonal high-producing clones secreting humanized-C2 monoclonal antibodies. The fluorescence labelling in semi-solid media also allows rapid visualization and discrimination of the rare high secretors from a majority of low producers (Caron et al 2009). …”

“…Theoretically, at equivalent cell densities, clones with higher the exterior fluorescence intensities should have higher concentration of antibodies being secreted. Furthermore, it has been reported that secreted recombinant protein levels correlated well with their fluorescence intensities (Caron et al 2009). However, our results show that at equivalent cell densities of 3 x 10 6 cells/ml, the exterior fluorescence intensities and antibody productivity or our humanized cells does not correlate well for clones above 1000 FU.…”