TBPP_coagulation factors

X-linked recessive hereditary bleeding disorders, A: deficiency of Factor VIII, B: deficiency of Factor IX, severe form: spontaneous bleedings, joint bleedings, treatment with substitution therapy: plasma or recombinant factor, IV 2-3 times per week, normal life expectancy and improved quality of life

most severe complication in treatment, development of antibodies against substituted factor (10-20 days), high risk of bleeding, acute treatment with bypass products (activated prothrombin factor concentrate, recombinant FVII) or immune tolerance induction: high doses of FVIII.

congenital deficiency/reduction of VWF, complex disease, most common coagulation disorder, classification: 1. quantitative reduction, 2. qualitative defects, 3. complete deficiency, treatment with plasma derived VWF concentrates.

no real treatment, cryoprecipitate in 60's, freeze-dried concentrates in 70's, virus inactivation in 80's, recombinant factors in 90's, today improved recombinant factors (longer half life)

dissolution of cryoprecipitate --> adsorption on Al(OH)3 (2x) --> stabilization --> pasteurization --> heating for 10h at 60°C --> anion exchange chr. --> dialysis --> virus filtration --> sterile filtration --> filling and lyophilisation. Up to now: 200T cryoprecipitate, 375g FVIII

DNA construct which codes for FVIII, transfection of DNA in eucaryontic cells (selection, protein secretion, posttranslational modifications), fermentation, purification, characterization (biological activity, pharmacokinetics). Fermentation: mammalian cell expansion, continous harvest, sensitive products. purification: different chr., virus inactivation, sterile bulk production, fill and freeze drying: single use vials, different strength. product release: assays, chemical tests, endotoxin/sterility, characterization

immunogenicity, extended treatment interval, non-IV application, price

beneficial to patients: less frequent injections, improved compliance, may enable prophylaxis. Technologies: sustained delivery, chemical or genetic modification, fusion with carrier proteins (Fc, albumin). Challenge: chronic therapy <--> acute therapy, half-life extension <--> biological activity, safety <--> improved therapy

albumin: naturally long half-life (20 days), highly abundant , carrier protein, molecular structure. DNA construct that codes for both proteins, expressed as single recombinant molecule, transfection into eukaryotic cells: selection, secretion of protein, post-translational modification

Factor IX-Albumin fusion protein. First fusion: specific activity significantly reduced. Second experiment: albumin fused to C-terminus of FIX, short linker between both containing glycine and serine, expression in CHO cells, PK-study in rats, half-life extension worked. Advanced concept: rIX-FP with cleavable linker derived from FIX activation region, activation by cleavage (by FXIa). Advantage: increased activity, activated FIX molecules not show extended half-life, disadvantages: albumin with linker fragment in circulation.

T-cell epitopes, T-cell activation triggers immune response, T-cell receptor and MHCII binding of neosequences (between protein sequences)

3 different approaches applied (8/51/14 MHC II allele), no/weak binding to MHCII allele, very low potential immunogenicity, studies in humans (prophylaxis)

approved in US, Canada, Europe (Switzerland), Japan. Significant improvement: dosing interval every 2 weaks, benefits for Hemophilia B patients