Donogh P. O'Brien

High-level production of human blood coagulation factors VII and XI using a new mammalian expression vector.

Recombinant human proteins are generally recovered in low yields from mammalian tissue culture following transfection with commercially available vectors. We have constructed a novel vector containing both the neomycin-resistance-encoding gene (neo) as a dominant selectable marker, and the dihydrofolate reductase-encoding gene (DHFR) to enable amplification of transfected DNA followed by stable expression in mammalian cell lines. Levels of 5 micrograms/ml of the coagulation proteins, factor VII (FVII) and factor XI (FXI), have been achieved in serum-free media. N-terminal sequencing of the purified proteins, and of their separated chains after proteolytic activation, demonstrated correct processing of the recombinant products. In addition, the ratios of clotting activity to antigen for each are close to unity, and the recombinant and plasma-derived proteins had identical mobilities upon electrophoresis in the presence of SDS. The vector described will be of use for the synthesis of recombinant proteins, both wild-type and variants produced by site-directed mutagenesis, especially where complex post-translational modification of the protein makes it essential to use mammalian cells.

Immunologic studies of factor VIII coagulant activity (VIII:C) 2. Factor VIII in selected vertebrates

Cross reactive antigens to factor VIII have been measured in a range of vertebrate phyla. VIII coagulant antigen (VIII:CAg) measured using a human antibody was in general, much lower than reported values of VIII coagulant activity (VIII:C) except in simians and the guinea pig. The dose response curve for the assay was parallel in all cases. Factor VIII-related antigen (VIIIR:Ag) measured using rabbit antibody to human VIIIR:Ag was low or absent in most species except simians and the dose response curve was non-parallel to the human standard except with goat plasma. Avians lacked cross-reactive material.

Affinity Depletion and Affinity Purification of Human Factor IX by Monoclonal Antibodies

Abstract Monoclonal antibodies to human coagulation factors are of potential use in the characterisation, detection and purification of these proteins. In addition, monoclonals could be used to affinity deplete specific factors from plasma to produce deficient substrate for use in one-stage coagulation assays. We have raised four monoclonal antibodies to human factor IX (RFF-IX/1, RFF-IX/2, RFF-IX/3 and RFF-IX/4) and have assessed their suitability for affinity depletion and affinity purification of factor IX. The factor IX deficient plasma so produced is capable of being used as substrate in a one-stage coagulation assay for factor IX and performs as well as does severe Christmas disease plasma. In addition, we have used one of the monoclonal antibodies to obtain highly purified factor IX from factor IX concentrate in a one-step purification procedure.