Elimination of ghost peaks by optimization of anion exchange chromatography method for determination of gamma-carboxyglutamic acid (Gla)-domainless impurity in recombinant activated clotting factor VII drug products.

Secreted recombinant activated clotting factor VII activated (rFVIIa) in cell culture media missing gamma-carboxyglutamic acid (Gla) domain as a result of failure in gamma-carboxylation or cell lysis is called Gla-domainless impurity which has less negative charge compared to native rFVIIa. Based on risk assessment, this type of impurity is considered as critical drug product quality attribute of rFVIIa and its quantitative analysis in product batches is a critical issue in quality control laboratories. Analysis of Gla-domainless impurity is accomplished by Strong Anion Exchange Chromatography (SAX) in recombinant factor VIIa using Tris and Bis-Tris propane salt buffers as equilibrating buffers and high concentration ammonium acetate as an eluent. Appearance of ghost peaks with notable intensity during elution time of Gla-domainless impurity caused distortion of the related peak and interference with robust and accurate quantification of this impurity. Subsequently, the ghost peak was analyzed by LC-ESI-MS to determine the structure which showed the m/z values at 905.27, 623.53 and 341.60 and 563.73. To find the source of these ghost peaks, quality of water, buffer salts and Chelex-100 together with ionic strength of mobile phase A (addition of 25 mM NaCl) were considered as affecting parameters and several experiments designed with DOE software to optimize the best condition of highest quality the method with lowest signal of ghost peak noises. By interpretation of DOE result, it is concluded that high grade water and buffer salt along with high quality Chelex-100 resins are important factors to achieve a method with lowest ghost peaks. However, addition of 25 mM NaCl to mobile phase A with either lower quality buffer salts or lower water grade yields high quality chromatogram peak with acceptable ghost peaks. LC/MS analysis indicates that macrostructures of Bis-Tris propane made up as a result of hydrogen bonds with each other or Tris molecules can be the source of ghost peaks.

New perspectives in hemophilia treatment.

A variety of factor concentrates are currently available for replacement therapy for patients with hemophilia. These differ by several parameters, including source (pooled from pooled blood vs recombinant), purity, pathogen inactivation, and by the presence or absence of extraneous proteins such as albumin. The choice of replacement product reflects both safety issues of pathogen transmission or inhibitor development, and personal preferences of the patient and the physician. In general, currently available products are viral pathogen-free, although there is debate about the risk of transmission of parvovirus B19 and prion pathogens. Because of this very small risk, recombinant factor is the treatment of choice in previously untreated patients. In addition, a subset of concentrates contain factor that is activated during manufacture, yielding activated products that can be used in the treatment of patients with inhibitors. Such activated products, especially recombinant factor VIIa (rFVIIa), have also acquired several off-label indications in the management of bleeding in non-hemophiliac patients. The management of hemophilia patients with inhibitors is an ongoing challenge. Immune tolerance induction using a desensitization technique is successful in up to 90% of patients with alloantibodies against factor VIII, with greatest success seen in patients with low titer inhibitors who are treated soon after detection of an alloantibody and in whom treatment includes administration of immunosuppression along with repeated infusions of high titer concentrates. Such therapy is less successful in patients with factor IX alloantibodies. Non-hemophiliac patients with acquired inhibitors represent a unique patient population that requires special management. These patients have a mortality rate that approaches 25% because of the association of acquired inhibitors with severe bleeding complications, occurrence in a largely elderly population, and the frequent presence of an underlying, often serious, primary medical condition. Treatment consists of immunosuppression with steroids, chemotherapy, or intravenous immunoglobulin. Recent studies using rituximab for selective B-cell depletion in these patients have been very promising, although prospective controlled studies have not yet been performed. Finally, although hemophilia A and B appear to be ideal diseases to target with gene therapy approaches, the promise of this therapy remains to be realized.

New products for managing inhibitors to coagulation factors: a focus on recombinant factor VIIa concentrate.

The treatment of alloantibody and autoantibody inhibitors directed against the factor VIII coagulant protein is one of the most challenging and expensive problems in hematology. Because the currently available plasma replacement products used in this context do not control the bleeding complications in all patients, and because of the usual emergent quality of the bleeding complications, there has been a definite need to have a uniformly reliable product for instant use, which possesses a high degree of hemostatic reliability and safety. The recent introduction of recombinant factor VIIa (rFVIIa) has been a welcome addition to the pharmacologic armamentarium for the treatment of neutralizing antibodies against coagulation factors. The mechanisms of action of rFVIIa have also been interesting and have provided insight into how the coagulation pathway accomplishes adequate hemostasis. This review will discuss this new medication and place into the context of coagulation inhibitor therapy.

Recombinant factor VIIa for patients with inhibitors to factor VIII or IX or factor VII deficiency.

Inhibitors to factor VIII (FVIII) or IX (FIX) in patients with haemophilia A or B create a challenging problem for the treatment of these patients. Recombinant FVIIa (rFVIIa; NovoSeven, Novo Nordisk A/S, Bagsvaerd, Denmark) is a realistic treatment option, owing to its specific mode of action and lack of immunogenicity. This was a multicentre, open-label, compassionate-use trial in patients with severe haemophilia A (FVIII:C < 1%) or B (FIX:C < 1%) with inhibitors, acquired antibodies to FVIII or FIX, or FVII deficiency (FVII:C < 5%), for whom alternative therapies had failed or were contraindicated. Patients received rFVIIa treatment for life- or limb-threatening bleeding episodes or for coverage during essential surgery. The mean rFVIIa dose was approximately 90 microg kg-1 for haemophilia A/B and acquired inhibitor patients, and 25 microg kg-1 for FVII-deficient patients. Efficacy data for 67 treatment episodes (45 bleeding episodes, 22 surgical procedures) are presented; seven patients were treated for a concurrent serious bleeding episode and surgical procedure. At the end of treatment, rFVIIa was effective or partially effective in 85% of serious bleeding episodes. During surgery, bleeding was assessed as none or less than or equivalent to normal in 91% of surgical procedures; postoperatively, 91% of procedures were associated with no or minimal oozing. During 60 separate treatment episodes, 26 adverse events (22 nonserious, four serious) were reported in 15 patients, during 17 bleeding episodes or surgical procedures. Only 10 were considered as having a possible, probable, or unknown relationship with rFVIIa; of these, fever (n=2) and thrombophlebitis (n=3) were the most common. There was no evidence of disseminated intravascular coagulation. In conclusion, rFVIIa is an effective, well-tolerated treatment for serious bleeding episodes and bleeding associated with surgical procedures in patients with severe haemophilia A/B with inhibitors, acquired inhibitors, or FVII deficiency.