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.

Figure 1. High-throughput sorting of the highest protein-productive cell by a transiently protein-anchored system.

(A) Strategy and organization of the transiently protein-anchored system. (B) Schematic representation of the gene construction of the transiently protein-anchored system using anti-EGFR antibody as an example. The construction includes, from N to C termini, an immunoglobulin leader sequence (LS), an HA epitope, the anti-EGFR antibody fragment, the furin cleavage site (RAKR), and the immunoglobulin C2-type extracellular-transmembrane-cytosolic domains of murin B7-1 antigen (B7).

Figure 2. Furin inhibitor-mediated switch of secreted αEGFR Ab and anchored αEGFR Ab-RAKR-B7.

(A) HEK-293/αEGFR cells were treated with 20 µM furin inhibitor for 24 h, and HEK-293 cells were used as a negative control. Cultured medium was coated on 96-well plates and were detected by ELISA using HRP conjugated goat anti-human IgGFcγ antibody. Bars, SD. (B) HEK-293 cells (filled histogram); HEK-293/αEGFR cells (solid line) and HEK-293/αEGFR cells treated with furin inhibitor (20 µM) (dashed line) were analyzed by flow cytometry using a specific antibody to the human IgGFcγ to assess the expression of membrane-anchored αEGFR Ab-RAKR-B7. (C) The cultured medium and cell lysate harvested from HEK-293 cells, HEK-293/αEGFR cells, and HEK-293/αEGFR cells treated with furin inhibitor (2 or 50 µM) were analyzed by western blotting using HRP conjugated goat anti-human IgGFcγ antibody. The clear bands appearing at 55 KD in lanes 2 and 3 correspond to the secreted αEGFR Ab heavy chain, and the clear bands appearing at 95 KD in lanes 7 and 8 correspond to the membrane-anchored αEGFR Ab heavy chain-RAKR-B7. The bands appearing at 80 KD in lanes 7, 8, and 9 correspond to the αEGFR Ab light chain-2A-heavy chain, and the bands appearing at 120 KD in lanes 7, 8, and 9 correspond to the αEGFR Ab light chain-2A-heavy chain-RAKR-B7.

Figure 3. Correlation between the secreted αEGFR Ab and the membrane-anchored αEGFR Ab-RAKR-B7.

(A) The HEK-293/αEGFR cells with high (green), medium (orange), or low (blue) expression levels of anchored αEGFR Ab-RAKR-B7 were sorted by flow cytometry. Original populations or sorted clones of HEK-293/αEGFR cells were confirmed by first being treated with furin inhibitor (20 µM), and then through analysis by flow cytometry using FITC conjugated goat anti-human IgGFcγ antibody. (B) The cultured media of sorted HEK-293/αEGFR cells were collected and analyzed by ELISA using HRP conjugated anti-human IgGFcγ antibody. Bars, SD.

Figure 4. Calculation of the correlation coefficient between the secreted αEGFR Ab and the membrane-anchored αEGFR Ab-RAKR-B7.

Each dot represents a monoclonal cell isolated from HEK-293/αEGFR cells. The cultured medium was collected from each clone and the secretion level of αEGFR Ab was determined by ELISA; the monoclonal cells were treated with furin inhibitor (20 µM) and analyzed by flow cytometry. The Y-axis represents the titer of secreted αEGFR Ab in the absence of furin inhibitor. The X-axis represents the mean relative fluorescent intensity of membrane-anchored αEGFR Ab-RAKR-B7 stained with FITC conjugated anti-human IgGFcγ antibody. Collectively, the titer of secreted αEGFR Ab and the surface expression level of anchored αEGFR Ab-RAKR-B7 reveal a highly positive correlation (R² = 0.9165).