Viral safety of B-domain deleted recombinant factor VIII.

The possible transmission of blood-borne pathogens has been the impetus behind the development of recombinant products formulated in the absence of human-derived components. The viral safety of Chinese hamster ovary (CHO)-cell-based pharmaceuticals is well established. Over 100 million infusions have been administered without a single known incident of CHO-related viral transmission. The manufacturing process for B-domain deleted recombinant factor VIII (BDDrFVIII) builds on this safety record by using a state-of-the-art multitiered approach to viral safety. This approach includes: (1) extensive testing of the CHO cells used to produce BDDrFVIII; (2) routine viral monitoring of the cell culture production process; (3) a purification process in which a specific viral inactivation procedure has been included; (4) a final formulation that does not incorporate human albumin as the stabilizer; and (5) a thorough validation of the viral inactivation and removal capacity of the purification process. This multifaceted viral safety program offers the hemophilia community a factor VIII product with an exceptional degree of viral safety.

Immobilization and affinity purification of recombinant proteins using histidine peptide fusions.

A gene fusion approach to simplify protein immobilization and purification is described. A gene encoding the protein of interest is fused to a gene fragment encoding the affinity peptide Ala-His-Gly-His-Arg-Pro. The expressed fusion proteins can be purified using immobilized metal affinity chromatography. A vector, designed to ensure obligate head-to-tail polymerization of oligonucleotide linkers was constructed by in vitro mutagenesis. A linker encoding the affinity peptide, was synthesized and polymerized to two, four and eight copies. These linkers were fused to the 3' end of a structural gene encoding a two-domain protein A molecule, ZZ, and to the 5' end of a gene encoding beta-galactosidase. Fusion proteins, of both types, with zero or two copies of the linker showed little or no binding to immobilized Zn2+, while a relatively strong interaction could be observed for the fusions based on four or eight copies of the linker. Using a pH gradient, the ZZ fusions were found to be eluted from the resin at different pHs depending on the number of the affinity peptide. These results demonstrate that genetic engineering can be used to facilitate purification and immobilization of proteins to immobilized Zn2+ and that the multiplicity of the affinity peptide is an important factor determining the binding characteristics.