Monitoring once-weekly recombinant factor IX prophylaxis in hemophilia B with thrombin generation assay and factor IX activity

2017 ◽  
Vol 39 (4) ◽  
pp. 359-368 ◽  
Author(s):  
V. Nummi ◽  
A. Jouppila ◽  
R. Lassila
Keyword(s):  
Thrombin Generation ◽  
Factor Ix ◽  
Hemophilia B ◽  
Thrombin Generation Assay ◽  
Recombinant Factor Ix

Impact of Factor IXa Content On Function, Safety and Efficacy of Recombinant Factor IX Products.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2233-2233
Author(s):  
Wilfried Auer ◽  
Hanspeter Rottensteiner ◽  
Gerald Schrenk ◽  
Werner Hoellriegl ◽  
Alexandra Schiviz ◽  
...  
Keyword(s):  
Factor Ix ◽  
Hemophilia B ◽  
In Vitro Assays ◽  
Safety And Efficacy ◽  
Clinical Dose ◽  
Thrombin Generation Assay ◽  
Recombinant Factor Ix ◽  
Thrombogenic Potential ◽  
Individual Scores

Abstract Abstract 2233 Baxter has developed a new recombinant factor IX (rFIX) product (BAX 326) for treating patients with hemophilia B. The aim of the presented studies was to evaluate the function, safety and efficacy of BAX 326 compared to a commercially available rFIX regarding differences in activated FIX (FIXa) content. The FIXa concentration of BAX 326 was about 10 fold less than that of a commercial rFIX product. To address the influence of FIXa on functional FIX in vitro assays, the FIXa concentration of BAX 326 was increased. Samples were analyzed by one-stage clotting assay, non-activated partial thromboplastin time assay and thrombin generation assay. In all three assays, FIXa affected the potency determination for FIX, indicating that rFIX products should preferentially contain a low FIXa content. The thrombogenic potential of BAX 326 was assessed in rabbits using a modified Wessler Test at 750 IU/kg (10-fold human clinical dose). No thrombogenic potential was observed with BAX 326 (individual scores of 0), whereas the mean score for the commercially available rFIX was 0.5. After increasing the FIXa concentration of BAX 326 to equalize it with the commercially available rFIX, a mean score of 0.42 (individual scores 0 – 0.5) was determined. These data strongly suggest that the differences in preclinical thrombogenicity were caused by the higher FIXa content of the commercially available rFIX, thereby confirming earlier findings [1]. Efficacy of the rFIX products was studied in hemophilia B (FIX ko) mice that received prophylactic treatment with 75 IU/kg of BAX 326 or the commercially available rFIX. Treated animals were analyzed in two primary pharmacodynamic models: a carotid occlusion model and one using thrombelastography. In both models, results for the two groups were similar, demonstrating that despite its lower FIXa content, BAX 326 was as efficacious as a commercially available rFIX at 75 IU/kg (p<0.0076). Disclosures: Auer: Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Schrenk:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment. Schiviz:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.

The Effect of Factor IXa on Thrombin Generation Activity Determination: rFIXFc Vs. BeneFIX®

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2266-2266
Author(s):  
Yang Buyue ◽  
Ekta Seth Chhabra ◽  
Li Wang ◽  
Jurg M Sommer
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Time Course ◽  
Drug Product ◽  
Factor Ix ◽  
One Stage ◽  
Thrombin Generation Assay ◽  
Factor Ixa ◽  
Recombinant Factor Ix

Abstract Abstract 2266 Recombinant Factor IX Fc fusion (rFIXFc) was designed to be a long-lasting version of recombinant FIX that has the potential to provide less frequent dosing. This may be applicable to prophylaxis and on-demand therapy of hemophilia B and for control of bleeding during surgery. The potency of the rFIXFc drug product, as well as the commercially available recombinant factor IX, BeneFIX®, is assigned by a one-stage clotting assay. The thrombin generation assay (TGA), a global hemostasis assay that monitors the amount of active thrombin produced in patient plasma after recalcification, represents a useful indication in the evaluation of coagulation capacity of hemophilic plasma. When equal units of rFIXFc and BeneFIX, as determined by the one stage assay, were spiked into hemophilic plasma and their coagulation capacity was assessed by the TGA, BeneFIX generated 2-fold higher peak thrombin and significantly left-shifted thrombin curve relative to rFIXFc in the presence of limiting tissue factor and 4 μM phospholipids. In an assay control without tissue factor triggering, BeneFIX demonstrated considerable thrombogenic activity, whereas rFIXFc was essentially inactive. Since BeneFIX was observed to have a markedly higher level of FIXa impurity than rFIXFc in a factor IXa ELISA, and since factor IXa protease is known to be highly thrombogenic in an in vivo model, we hypothesized that the enhanced in vitro thrombin generation profile of BeneFIX may be due to the presence of excess factor IXa. BeneFIX was incubated overnight with a serine protease active site blocker, EGR-chloromethyl ketone, and dialyzed by extensive buffer exchange. The FIXa-blocked BeneFIX showed very similar thrombin generation profile (ETP, peak thrombin, time course and slope) to rFIXFc, confirming the role of FIXa in thrombin generation by BeneFIX. To quantify the amount of active factor IXa in BeneFIX and rFIXFc, a plasma-derived factor IXa (pFIXa) standard curve was constructed by spiking increasing concentrations of factor IXa (0–100 pM) into human factor IX-deficient plasma in the presence of 4 μM phospholipids. Prior to starting the measurement, 5 nM thrombin was added to the assay in order to improve sensitivity. A dose response was observed with a detection limit as low as 0.5 pM pFIXa in FIX-deficient plasma. BeneFIX, FIXa-blocked BeneFIX and rFIXFc of equal potency (1 IU/mL by the one-stage clotting assay) generated thrombin responses comparable to 20 pM, 1 pM and 2 pM pFIXa, respectively, indicating the amount of FIXa present in each FIX product. In a regular thrombin generation assay triggered with limiting tissue factor, 1 IU/mL rFIXFc supplemented with 20 pM pFIXa demonstrated an equal peak thrombin and velocity index to 1 IU/mL BeneFIX. These data suggest that: 1) Minor amounts of FIXa protease in a FIX drug product (0.1%) can trigger significant thrombin generation in global hemostasis assays 2) Thrombin generation assay could be used to evaluate FIXa level in FIX products with high sensitivity (0.5 pM FIXa per IU/ml FIX) 3) the higher peak thrombin and shortened time course in the thrombin generation profile for BeneFIX relative to rFIXFc is caused entirely by the presence of factor IXa in the drug product 4) Discounting the rFIXa impurities in these drug products, BeneFIX and rFIXFc have equivalent in vitro thrombin generation activity per unit of FIX activity. Disclosures: Buyue: Biogen Idec Hemophilia: Employment. Seth Chhabra:Biogen Idec Hemophilia: Employment. Wang:Biogen Idec Hemophilia: Employment. Sommer:Biogen Idec Hemophilia: Employment.

scholarly journals Treatment of hemophilia B: focus on recombinant factor IX

2013 ◽  
pp. 33 ◽  
Author(s):  
Massimo Franchini ◽  
Frattini ◽  
Crestani ◽  
Sissa ◽  
Bonfanti
Keyword(s):  
Factor Ix ◽  
Hemophilia B ◽  
Recombinant Factor Ix

Binding To Phospholipid Protects Factor VIII From Inhibition By Human Antibodies

1981 ◽  
Author(s):  
T W Barrowcliffe ◽  
E Gray ◽  
G Kemball-Cook
Keyword(s):  
Factor Viii ◽  
Thrombin Generation ◽  
Total Phospholipid ◽  
Fluid Phase ◽  
Clinical Applications ◽  
Factor Ix ◽  
Two Stage ◽  
Human Antibodies ◽  
Thrombin Generation Assay ◽  
Detectable Antigen

Previous studies with activated Factor IX concentrates have suggested that they may contain a form of Factor VIII clotting activity (VIII:C) which is partly protected from inactivation by antibodies. A possible mechanism for such protection is binding to phospholipid. The interaction between Factor VIII, phospholipid and human antibodies to Factor VIII was studied by a two-stage clotting assay, and by a fluid-phase immunoradiometric assay for Factor VIII clotting antigen (VIII C:Ag).In the two-stage thrombin generation assay, Factor VIII:C was rapidly destroyed by human antibodies, even in the presence of optimal phospholipid. However, preincubation of Factor VIII with phospholipid before addition of antibody protected the Factor VIII from inactivation, resulting in the production of much more thrombin.In assays of VIII C:Ag, pre-incubation of Factor VIII with phospholipid before addition of labelled antibody reduced the amount of detectable antigen. The reduction was greater with increasing phospholipid concentration, up to 60% of the original antigen being ‘lost’ at a total phospholipid concentration of around 250 μg/i.u.These results suggest that human antibodies to Factor VIII are directed largely at its phospholipid binding site. The protection of Factor VIII from inactivation by complexing with phospholipid could have important clinical applications in treatment of haemophiliacs with inhibitors.

scholarly journals Favorable pharmacokinetics in hemophilia B for nonacog beta pegol versus recombinant factor IX-Fc fusion protein: A randomized trial

2019 ◽  
Vol 3 (2) ◽  
pp. 268-276 ◽  
Author(s):  
Carmen Escuriola Ettingshausen ◽  
Inga Hegemann ◽  
Mindy L. Simpson ◽  
Adam Cuker ◽  
Roshni Kulkarni ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Randomized Trial ◽  
Protein A ◽  
Factor Ix ◽  
Hemophilia B ◽  
Fc Fusion Protein ◽  
Recombinant Factor Ix

Biochemical Characterization of a Recombinant FIX Drug Candidate for Treatment of Hemophilia B

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4658-4658
Author(s):  
Peter Turecek ◽  
Susanne Vejda ◽  
Katalin Varadi ◽  
Hanspeter Rottensteiner ◽  
Ernst Boehm ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Drug Product ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Drug Candidate ◽  
Alternative Drug ◽  
Recombinant Factor Ix ◽  
Fermentation Technology

Abstract Abstract 4658 Human coagulation factor IX (FIX) is a vitamin-K-dependent coagulation factor whose absence or dysfunction causes hemophilia B. Treatment of hemophilia B is based on replacement therapy using highly purified FIX concentrates. Baxter has developed a recombinant factor IX for treating hemophilia B patients that is produced in a CHO cell-line using a serum and protein-free fermentation technology. The purification process avoids the use of immune-affinity chromatography and includes two viral reduction steps. The final drug product is formulated in the absence of proteins of animal or human origin. Baxter's recombinant FIX resembles commercially available rFIX in most characteristics with the exception of a significantly lower FIXa content, which might improve standardization compared to commercial rFIX products. Preclinical and clinical lots of rFIX were characterized with respect to their hemostatic potency, efficiency of activation by FXIa and FVIIa in the presence of tissue factor, and capacity to bind to phospholipid vesicles. Three lots of commercial rFIX with different potencies and one lot of a plasma-derived FIX product were included in the study. Similarity could be shown between the preclinical and clinical lots of rFIX in all these assays. Furthermore, the functional and biochemical characterization of Baxter's recombinant FIX showed that it resembles the recombinant comparator product. The phase I clinical trial which has been initiated will now have to show whether Baxter's rFIX can become an alternative drug candidate product for treating patients suffering from hemophilia B. Disclosures: Turecek: Baxter Innovations GmbH: Employment. Vejda:Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Boehm:Baxter Innovations GmbH: Employment. Reiter:Baxter Innovations GmbH: Employment. Kaliwoda:Baxter Innovations GmbH: Employment. Mundt:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

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