Characterization and viral safety validation study of a pasteurized therapeutic concentrate of antithrombin III obtained through affinity chromatography.

BACKGROUND AND OBJECTIVE: Antithrombin III (ATIII) concentrates are employed as therapy for congenital or acquired deficiencies. These concentrates are obtained from Cohn's fraction IV1. To improve yields, purity and safety, our group developed a procedure to obtain a pasteurized ATIII concentrate from the supernatant of Cohn's fraction II + III including a highly efficient heparin affinity chromatography purification and pasteurization as a viral inactivation step. DESIGN AND METHODS: Three steps of the manufacturing procedure (Cohn's fraction II + III precipitation, affinity chromatography and pasteurization) were selected to examine their efficacy in inactivating and/or removing the selected viruses. RESULTS: The industrial batches show a purity higher than 99% with approximately 95% native heparin binding ATIII. Only albumin and IgG could be detected at trace levels (0.07% and 0.16% of the total protein present, respectively). The specific activity of the product was approximately 6.65 IU/mg protein. Five viruses were spiked into the manufacturing starting materials and samples were collected at various points to determine the infection level of virus. The study showed a reduction factor (log 10) > or = 11.7 for HIV-1; > or = 8.1 for bovine herpes virus (analyzed as a model for herpes and hepatitis B viruses); > or = 8.1 for bovine diarrhea virus (model for hepatitis C and G) and > or = 6.0 for encephalomyocarditis virus (model for hepatitis A and other non-enveloped viruses). INTERPRETATION AND CONCLUSIONS: No biochemical alterations of the ATIII were detected in the final product. A high viral elimination capacity of the production process was demonstrated. So far, more than 32 million units of ATIII have been transfused in the form of this therapeutic concentrate without any detected seroconversion.

[Development and characterization of a high-purity human factor VIII concentrate submitted to 2 specific virus inactivation treatments (FANDHI)]. / Desarrollo y caracterización de un concentrado de factor VIII humano de alta pureza, sometido a dos tratamientos específicos de inactivation viral (Fanhdi).

AIM: To develop a therapeutic human high purity FVIII concentrate, treated with two documented and complementary specific inactivation methods, for the treatment of haemophilia A. MATERIAL AND METHODS: Cryoprecipitate was obtained from human plasma, normal for ALT levels and negative for HBsAg and antibodies to HIV and to HCV. The cryoprecipitate was resuspended and purified with PEG. The PEG precipitate was resuspended and treated with TNBP/Polysorbate 80. The mixture was processed by heparin affinity chromatography. The eluate was concentrated and precipitated with glycine and salts. After resuspension, stabilizers were added and the solution was sterile filtered, dispensed in vials and lyophilized. These final vials were treated at 80 degrees C for 72 hours: FVIII:C, vWF:RCo, vWF:Ag, vWF multimeric structure and the concentration of other plasmatic proteins were analyzed. RESULTS: FVIII: C specific activity was between 1000 and 3000 U/mg (after elimination of vWF, present as a stabilizer and before the addition of human pasteurized albumin). vWF:RCo activity (0.7 U vWF:RCo/U FVIII:C) and the multimeric structure of vWF showed a good degree of conservation. Other plasmatic proteins analyzed were undetectable or at trace amounts. No prekalllikrein, kallikrein or activated coagulation factors activity could be detected. CONCLUSION: The FVIII concentrate described shows a high degree of purity and stability, which makes it very suitable for haemophilia A treatment.

[Evaluation of viral safety of a high-purity human factor VIII concentrate submitted to 2 specific virus inactivation treatments (FANDHI)]. / Estudio de validación de la seguridad viral de un concentrado de factor VIII humano de alta pureza, sometido a dos tratamientos específicos de inactivación viral (FANHDI).

AIM: To perform a validation study of the production process of a human high purity FVIII concentrate, obtained by affinity chromatography and treated with solvent-detergent and 80 degrees C, 72- hour dry heating in the final vial, in order to demonstrate its viral safety. MATERIAL AND METHODS: The ability to inactivate or eliminate viruses was studied in the steps of PEG precipitation, solvent-detergent treatment (6 h 25 degrees C), affinity chromatography and lyophilization plus heating 80 degrees C for 72 h. HIV and models for hepatitis A, B and C, as well as a model for parvovirus B-19 were employed. The experiments were carried out by spiking the samples at each step with 10% of their volume with the highest titer available virus culture. The samples were processed under validated conditions (mimicking the industrial process) and the residual infectivity was determined (as well as p24 antigen and reverse transcriptase for HIV at the solvent-detergent step). RESULTS: No residual infectivity could be detected for enveloped viruses (HIV and models for hepatitis B and C) after the first minutes of solvent-detergent treatment, which lasts 6 hours. Lyophilization followed by heating 80 degrees C for 72 hours caused complete disappearance of infectivity for the models of hepatitis A and C, before 24 hours of a treatment which lasts 72. Furthermore, lyophilization plus heating reduced infectivity for the models of hepatitis B and parvovirus B-19 by 3.4 and 4.1 logs, respectively. The affinity chromatography reduced infectivity by 7.6 logs for the model of hepatitis B and 2 logs for HIV. PEG precipitation also reduced the infectivity by 3.3 logs for the model of hepatitis A and by 1.2 logs for the model of parvovirus B-19. Taking the process as whole, the study showed cumulative reduction values between 5.3 and > 19 logs of the analyzed viruses. 25 million FVIII units have been transfused so far as FANHDI, with no seroconversion detected. Furthermore, no increase in FVIII inhibitor frequency has been described. CONCLUSION: The FVIII concentrate described shows outstanding viral safety characteristics. These data, together with the preliminary clinical experience after one year usage of the product, indicate that FANHDI is a suitable preparation for haemophilia A treatment.

Physico-chemical and analytical studies on plafibride.

Physico-chemical and analytical studies on N-2-(p-chlorophenoxy)-isobutyryl)-N'-morpholinomethylurea (plafibride, ITA 104) are reported. Besides the usual analytical data, spectral (UV, IR, NMR, MS) and chromatographic (HPLC, TLC) properties are described. As to chemico-analytical procedures, ITA 104 is detectable by titrimetric and spectralphotometric methods and by thin-layer chromatography.