Assessment of functional quality of Lenti-CMV-huVWF expressed by COS-7 cells or by CHO-K1 cells. (A) COS-7 cells were transfected with Lenti-CMV-huVWF or pNUT-VWF, and serum-free expression medium was harvested, pooled, and concentrated. (B) CHO-K1 cells were transduced with Lenti-CMV-huVWF, and serum-containing medium was harvested. VWF:Ag (▪), VWF:RCo (□), and VWF: CBA (▦) activities were determined in ELISA assays. VWF:RCo and VWF:CBA were determined by measuring the binding of VWF to rGPIbα in the presence of ristocetin and to human collagen type III respectively (A,B). VWF:RCo and VWF:CBA activities were absent in the serum-containing medium harvested from the nontransduced CHO-K1 cells. A human standard plasma pool was used as a reference (with VWF:Ag, WVF:RCo, and VWF:CBA all 1 U/mL). Data are mean ± SEM (n = 3). (C) Multimer analysis was conducted on 20 ng Lenti-CMV-huVWF present in the conditioned medium from transduced CHO-K1 cells (CHOK1-VWF) and on 60 ng VWF present in normal human plasma pool. Serum-containing medium (medium) was used as a negative control.