Ishaque Adiba, Thrift John, Murphy John E, Konstantinov K
Bayer Healthcare, Pharmaceuticals, Global Biological Development, Berkeley, CA 94701, USA.
J Biotechnol. 2008 Oct 10;137(1-4):20-7. doi: 10.1016/j.jbiotec.2008.07.1856. Epub 2008 Jul 25.
We have recently demonstrated an increase in recombinant factor VIII (rFVIII) secretion from BHK-21 cells (rBHK-21(host)) following an over-expression of the chaperone protein heat shock protein 70 (Hsp70) (rBHK-21(Hsp70)) 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-21(host), rBHK-21(Hsp70), and parental BHK-21 cells devoid of rFVIII expression (BHK-21(native)) 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-21(host) cells as the cell viability declined while the rFVIII cell surface staining increased. Conversely, rBHK-21(Hsp70) 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-21(host) cells and its reduction on the surface of rBHK-21(Hsp70) 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-21(native) batch cell cultures. However, PS exposure could not be identified to the same extent on rBHK-21(host) cells despite a similar decline in cell viability between rBHK-21(host) and BHK-21(native) batch cultures. Any exposed PS on rBHK-21(host) 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.
我们最近证明，由于凋亡细胞死亡受到抑制且重组因子VIII（rFVIII）的细胞表达增加，伴侣蛋白热休克蛋白70（Hsp70）过表达后，BHK - 21细胞（rBHK - 21（宿主））分泌的重组因子VIII（rFVIII）有所增加[Ishaque, A., Thrift, J., Murphy, J.E., Konstantin, K., 2007. 在工程化生产重组因子VIII的BHK - 21细胞中过表达Hsp70可促进抗凋亡并增强分泌。生物技术与生物工程。生物技术与生物工程。97, 144 - 155]。在本研究中，我们通过比较rBHK - 21（宿主）、rBHK - 21（Hsp70）和无rFVIII表达的亲本BHK - 21细胞（BHK - 21（天然））在分批细胞培养实验中细胞活力的变化以及凋亡过程中磷脂酰丝氨酸（PS）暴露程度，研究了rFVIII与细胞膜表面结合的差异。使用Zenon技术通过流式细胞仪Guava PCA分析对细胞表面和细胞内rFVIII进行双重染色。通过这种定量分析表明，随着rBHK - 21（宿主）细胞活力下降，细胞内rFVIII减少，而rFVIII细胞表面染色增加。相反，rBHK - 21（Hsp70）细胞培养物显示出更高的细胞活力和细胞内rFVIII，细胞表面rFVIII染色较少。荧光显微镜也证实了rFVIII与rBHK - 21（宿主）细胞表面结合的时间依赖性增加及其在rBHK - 21（Hsp70）细胞表面的减少。此外，更大的rFVIII细胞表面染色与BHK - 21（天然）分批细胞培养物表面可检测到的PS暴露增加相关。然而，尽管rBHK - 21（宿主）和BHK - 21（天然）分批培养物之间细胞活力有相似的下降，但在rBHK - 21（宿主）细胞上无法以相同程度鉴定到PS暴露。rBHK - 21（宿主）细胞上任何暴露的PS很可能被分泌的rFVIII掩盖，这类似于对活化血小板的影响，在活化血小板中也会发生PS的外化，并在凝血级联反应中作为FVIII激活的配体。综上所述，我们已经确定rFVIII在膜表面的隔离是rFVIII生产力的另一个潜在限制因素，并且在表达人HSP70的克隆中通过减少凋亡也可以缓解这一限制。
