Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A.

Factor VIII-specific affibodies were selected from phage displayed libraries constructed by combinatorial mutagenesis of an alpha helical bacterial receptor domain derived from staphylococcal protein A. Bead-immobilized recombinant human factor VIII (rVIII) (80 and 90 kDa chains) protein was used during competitive biopannings in the presence of free 80-kDa chain protein, resulting in the selection of several binders that showed dissociation constants (Kd) in the range 100-200 nM as determined by biosensor analyses. One variant (Z[rVIII:3], 90-kDa chain specific) was further characterized in small-scale affinity chromatography experiments, and showed efficient and selective recovery of biologically active rVIII from Chinese hamster ovary cell supernatant-derived feed stocks. The purity of the enriched rVIII was comparable with rVIII material purified by immunoaffinity chromatography using a 90-kDa chain-specific monoclonal antibody. Interestingly, epitope mapping showed that the monoclonal antibody and the affibody ligand competed for the same or at least overlapping epitopes on rVIII. In addition, the Z[rVIII:3] variant was produced by peptide synthesis with a C-terminal cysteine to enable directed coupling to solid supports. This 59-residue protein was analyzed by circular dichroism and showed a secondary structure content similar to that of the parental Z domain used as scaffold. In biosensor studies, the synthetic affibody was immobilized recruiting the C-terminal cysteine residue, and demonstrated to bind both recombinantly produced and plasma-derived factor VIII. From a secondary library, constructed by re-randomization of relevant positions identified after alignment of the first-generation variants, a panel of affinity-improved second-generation affibodies were selected of which one clone showed a dissociation constant (Kd) for rVIII of 5 nM. Several of these variants also showed higher apparent binding efficiencies towards rVIII when analyzed as immobilized ligands in biosensor experiments. Taken together, the results suggest that affibody ligands produced by bacterial or synthetic routes could be of interest as an alternative to monoclonal antibodies in purification processes or as diagnostic or monitoring tools.

The manufacturing process for B-domain deleted recombinant factor VIII.

The development of a cell bank used in the routine manufacturing of a B-domain deleted recombinant coagulation factor VIII (BDDrFVIII) molecule involved stable insertion of the human BDDrFVIII gene into Chinese hamster ovary (CHO) cells, selection of a cell line capable of expressing consistent levels of active BDDrFVIII, and the establishment of a cell bank. The manufacturing process begins with the culturing of CHO cells in large bioreactors. Product synthesis is initiated by altering the cell culture conditions, thereby arresting the cells in a stationary growth phase and inducing elevated expression of BDDrFVIII. Harvested culture medium is concentrated by chromatography and then purified through a series of four column chromatography steps and one solvent-detergent virus inactivation step. By eliminating the presence of human serum albumin in the final formulation, the BDDrFVIII-containing coagulant product meets with a high standard of safety against microbial and viral contamination. Extensive studies have shown that BDDrFVIII is a consistent, highly pure factor VIII for the treatment of patients with hemophilia A.

Effects of four treatment methods on social phobic patients not suitable for insight-oriented psychotherapy.

Forty-two social phobic men and women, rated unsuitable for insight-oriented psychotherapy, received one of four randomly assigned types of treatment for 3 months. All patients received basal therapy (B) in the form of standardized information, self-exposure instructions, and anxiolytic medication. One group received this treatment only, with monthly appointments. The others, in addition, received either therapist-directed prolonged exposure in vivo, in some cases supplemented with exposure in imagination (PE), dynamically oriented supportive therapy (ST) or relaxation therapy (R). There was a 9 months' follow-up period. The phobia variables were more improved in the PE and ST groups than in the R and B groups at the termination of treatment. Although improvement had deteriorated somewhat in the PE group during follow-up, the improvement in target phobia was better than in the other groups. There were almost no improvements in the R and B groups. ST and PE groups also showed improved social function while the global rating showed most improvement in the PE group. The B group was not improved at all and the R group only showed a short-lived drop in muscular tension.

Effects of four treatment methods on agoraphobic women not suitable for insight-oriented psychotherapy.

Seventy-three agoraphobic women rated unsuitable for insight-oriented psychotherapy, entered one of four randomly assigned types of treatment for 3 months. All patients received basal therapy in the form of standardized information, self-exposure instructions and anxiolytic medication. One group received this treatment only at monthly appointments (B). The others, in addition, received either therapist-directed prolonged exposure in vivo (PE), dynamically oriented supportive therapy (ST) or relaxation therapy (R). There was a 9-month follow-up period. All groups were about equally improved at the termination of treatment, with some advantage for PE and ST. At follow-up the ST group was more improved than the other groups, R was least improved, and PE showed some deterioration between the termination of treatment and the follow-up. ST, which took into account the overall adjustment as well as the symptoms, was thus the most successful treatment, but the superiority of the treatment did not manifest itself until at the end of the follow-up period.

Effects of three non-insight-oriented treatment methods on agoraphobic women suitable for insight-oriented psychotherapy.

Twenty-three agoraphobic women rated suitable for insight-oriented psychotherapy received one of three randomly assigned non-insight-oriented types of treatment for 3 months and the effect was followed up after 9 months. All patients received basal therapy (B) in the form of standardized information, self-exposure instructions and anxiolytic medication. One group received this treatment only with monthly appointments. The others, in addition, received either therapist-directed prolonged exposure in vivo (PE) or relaxation therapy (R). The effects of insight-oriented psychotherapy could not be studied due to the small numbers in this group. All groups showed clinically relevant improvement, which indicates a favourable prognosis of patients suitable for insight-oriented psychotherapy irrespective of mode of treatment. However, since the PE group was least improved in some neurotic symptoms and had two treatment drop-outs and two cases of symptom substitution this mode of treatment cannot be recommended for insight-suitable patients. R patients came out favourably, which was contrary to the poor outcome with this therapy in patients not suitable for insight-oriented psychotherapy.