High-Throughput Sorting of the Highest Producing Cell via a Transiently Protein-Anchored System

Abstract

Developing a high-throughput method for the effecient selection of the highest producing cell is very important for the production of recombinant protein drugs. Here, we developed a novel transiently protein-anchored system coupled with fluorescence activated cell sorting (FACS) for the efficient selection of the highest producing cell. A furin cleavage peptide (RAKR) was used to join a human anti-epithelial growth factor antibody (αEGFR Ab) and the extracellular-transmembrane-cytosolic domains of the mouse B7-1 antigen (B7). The furin inhibitor can transiently switch secreted αEGFR Ab into a membrane-anchored form. After cell sorting, the level of membrane αEGFR Ab-RAKR-B7 is proportional to the amount of secreted αEGFR Ab in the medium. We further selected 23 αEGFR Ab expressing cells and demonstrated a high correlation (R2 = 0.9165) between the secretion level and surface expression levels of αEGFR Ab. These results suggested that the novel transiently protein-anchored system can easily and efficiently select the highest producing cells, reducing the cost for the production of biopharmaceuticals.

Analysis of the immunoglobulin G (IgG) secretion efficiency in recombinant Chinese hamster ovary (CHO) cells by using Citrine-fusion IgG

“…As indicated by no correlation between the fluorescent intensities and the specific productivity ( Fig. 5b and e), it is difficult to obtain high producer cells by using the fluorescent intensity alone, although this was achieved by previous FACS studies (Meng et al 2000;DeMaria et al 2007;Sleiman et al 2008;Chuang et al 2014). The reason for this discrepancy might be explained by the isolation of single cells without synchronization of the cell cycle.…”

SPLICELECT™: an adaptable cell surface display technology based on alternative splicing allowing the qualitative and quantitative prediction of secreted product at a single-cell level

“…Even direct surface staining of the producing cells has been proposed, but the nature of the signal is not well understood and may not always be directly linked to the level of secretion. [32][33][34][35] Many of the limitations described above can be overcome by cell surface display of a membrane version of the target protein, for example, by linking the protein to the cell surface with a glycosylphosphatidylinositol anchor 36 or a transmembrane domain with a cleavable peptide, 8,37,38 so that the protein of interest is released after proteolytic treatment. The co-expression of an "anchor" protein at the cell surface in order to link the recombinant protein to the producing cell was recently described.…”

Development of a shear stress-free microfluidic gradient generator capable of quantitatively analyzing single-cell morphology

“…In the past two decades, high-throughput screening (HTS) and high-content screening (HCS) have become major landmarks in the field of drug discovery, leading to fast identification of new therapeutic molecules and novel genetic engineering strategies (Zhao et al 2015 ; Lovitt et al 2013 ; Carlson-Stevermer et al 2016 ; Macchi et al 2016 ). This has largely been accomplished by miniaturization and automation, for example by developing large multiwell plate-based screens (Nishihara et al 2016 ; Vrij et al 2016 ; Spencer et al 2016 ), customized biomolecule/cell arrays (Beachley et al 2015 ; Zhao et al 2015 ; Kwon et al 2011 ), cell sorting (Liu et al 2016 ; Stowe et al 2015 ; Chuang et al 2014 ) and microfluidics (Du et al 2016 ; Barata et al 2016 ). Microfluidics has made an important contribution to HTS and HCS methodologies by enabling experiments with small amounts of reagents and low cell numbers.…”