Cell surface staining of recombinant factor VIII is reduced in apoptosis resistant BHK-21 cells

We have recently demonstrated an increase in recombinant factor VIII (rFVIII) secretion from BHK-21 cells (rBHK-21host) following an over-expression of the chaperone protein heat shock protein 70 (Hsp70) (rBHK-21Hsp70) due to an inhibition of apoptotic cell death and an increased cellular expression of rFVIII [Ishaque, A., Thrift, J., Murphy, J.E., Konstantin, K., 2007. Over-expression of Hsp70 in BHK-21 cells engineered to produce recombinant factor VIII promotes resistance to apoptosis and enhances secretion. Biotechnol. Bioeng. Biotech. Bioeng. 97, 144–155]. In the present study we investigated the difference in adherence of rFVIII to the cell membrane surface by comparing changes in cell viability and extent of phosphatidylersine (PS) exposure in apoptosis between rBHK-21host, rBHK-21Hsp70, and parental BHK-21 cells devoid of rFVIII expression (BHK-21native) during batch cell culture experiments. The Zenon technique was used to double stain for cell surface and intracellular rFVIII using flow cytometric Guava PCA™ analysis. By this quantitative analysis intracellular rFVIII was shown to decrease in rBHK-21host cells as the cell viability declined while the rFVIII cell surface staining increased. Conversely, rBHK-21Hsp70 cell cultures displayed higher cell viability and intracellular rFVIII with less cell surface rFVIII staining. Time dependent increases of rFVIII adherence to the surface of rBHK-21host cells and its reduction on the surface of rBHK-21Hsp70 cells was also confirmed by fluorescence microscopy. Furthermore, greater rFVIII cell surface staining correlated with an increase in detectable PS exposure on the surface of BHK-21native batch cell cultures. However, PS exposure could not be identified to the same extent on rBHK-21host cells despite a similar decline in cell viability between rBHK-21host and BHK-21native batch cultures. Any exposed PS on rBHK-21host cells was most likely masked by secreted rFVIII, mimicking the effect on activated platelets where the externalization of PS also occurs, and serves as a ligand for FVIII activation in the blood coagulation cascade. Taken together we have identified that rFVIII sequestration on the membrane surface is another potential limitation to rFVIII productivity and one which can also be alleviated by reduction of apoptosis in a clone expressing human HSP70.