Inactivation and elimination of viruses during preparation of human intravenous immunoglobulin.

We report here the results of our evaluation of virus inactivation during the manufacturing steps of two intravenous immunoglobulin (IGIV) preparations. Virus inactivation and/or removal by processing steps, such as ethanol fractionation and polyethylene glycol precipitation, and deliberate virucidal steps, such as solvent/detergent treatment and pasteurization, were tested on a variety of human pathogenic and experimental model viruses, including human immunodeficiency, Hepatitis C, Mumps, Vaccinia, Chikungunya, Vesicular Stomatitis, Sindbis, and ECHO viruses. All viruses were successfully inactivated and/or eliminated by the processing steps studied. In some cases, however, multiple steps were required. We conclude that the incorporation of steps deliberately designed to inactivate or remove viruses during the production of IGIV provides an extra measure of viral safety.

Human coagulation factor IX: assessment of thrombogenicity in animal models and viral safety.

Thromboembolic complications associated with prothrombin complex concentrate treatment may be related to the high levels of factors II and X in these products. We report here results from preclinical safety studies with a human coagulation factor IX product (AlphaNine; Alpha Therapeutic Corp., Los Angeles, Calif.) that contains no detectable factor II or VII and less than 10 units of factor X/100 units of factor IX. This product was manufactured from virally inactivated factor IX complex with a barium citrate adsorption step followed by affinity chromatography yielding factor IX concentrate with a specific activity of about 86 factor IX units/mg protein. Electrophoresis and immunoblot analysis indicated that the factor IX represents about 65% of the protein in this product. The virus inactivation step incorporated into the manufacturing process (incubation with n-heptane at 60 degrees C for 20 hours) was shown to inactivate at least 8.6 logs of type 1 human immunodeficiency virus. The barium citrate adsorption and affinity chromatography steps were found to remove 2.0 logs of the marker virus, vaccinia, and the DEAE ion-exchange chromatography used to produce factor IX complex was found to remove 1.4 logs of the marker virus, Sindbis. Analysis of three separate manufacturing lots with the polymerase chain reaction revealed no evidence of hepatitis C virus. The purified factor IX was nonthrombogenic when tested at doses of 450 units/kilogram in a rabbit stasis (Wessler) model, whereas the prothrombin complex concentrates were found to be thrombogenic at doses of less than 50 units/kg. There was no evidence of DIC in a porcine model after infusion of 200 units/kg of coagulation factor IX, as manifested by negative fibrin monomer tests, the absence of fibrin in blood vessels at autopsy, little or no change in prothrombin times and partial thromboplastin times, and only moderate decreases in platelet levels after infusion.

The fibrinolytic potential of the normal primate following the generation of thrombin in vivo.

Parameters of the fibrinolytic system were studied in a primate model where the generation of thrombin was promoted in vivo. The procoagulant stimulus used was a combination of human factor Xa in combination with phosphatidylcholine/phosphatidylserine lipid vesicles (PCPS) as the source of coagulant active phospholipid. The dosage of each component was formulated to provide a gradation of thrombin generating potential assessed prior to in vivo study in an in vitro clotting assay. These ranged from 25.25-36.60 pMole/kg (factor Xa) and 18.85-56.30 nMole/kg (PCPS). In each case, the ratio of the dose of factor Xa/PCPS was maintained at 0.65 (pMole factor Xa/nMole PCPS). Individual dosage combinations producing recalcification clotting times in vitro of 15, 20, 25 and 30 s were used in detailed in vivo studies. Previous studies in dogs had confirmed the thrombin generating potential of factor Xa/PCPS infusions and demonstrated an associated activation of protein C and increased fibrinolytic activity. This has now been extensively characterized in the chimpanzee as follows: 10 min after the infusion of the highest dose (36.6 pMole factor Xa/56.3 nMole PCPS kg bodyweight), the level of circulating t-PA had risen to 900 ng/ml (antigen), 885 IU/ml (functional). Dosage was observed with the lowest dose of 12.25 pMole factor Xa and 18.85 nMole PCPS being associated with relatively minor increases in circulating t-PA activity. There were no changes in u-PA at any dosage during the full time course of the experimental period (90 min). Plasminogen activation was also apparent with alpha-2 antiplasmin levels falling to 30-40% of pre-infusion levels at the highest dosages.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of human viral disease transmission through plasma products.

Alpha's Wet Heat-Treatment process is being applied to both Factor VIII (AHF) and Factor IX Complex (PTC). Twelve hemophilia A, five hemophilia B, and one von Willebrands patient have been followed for at least 6 months for evidence of non-A, non-B hepatitis. No ALT elevations were seen in the hemophilia B patients. There have been four cases of ALT elevation, three in hemophilia A patients and one in the von Willebrand's patient. A subset of these patients have been followed for over one year for anti-HTLV-III status. No patient, either hemophilia A, hemophilia B, or von Willebrand's seroconverted to anti-HTLV-III positive status. Intravenous gamma globulin was studied in 11 normal patients given a single infusion and in 23 immune deficient patients with multiple infusions and evaluations of liver enzymes over a two year period. No elevated ALT or AST values were seen in either group.

Evaluation of two viral inactivation methods for the preparation of safer factor VIII and factor IX concentrates.

We report here the results of our evaluation of two procedures to eliminate viruses in factor VIII and factor IX coagulation factor concentrates. Both procedures were equally effective in the in vitro destruction of marker viruses. However, in a controlled infectivity test in chimpanzees, treatment at 60 degrees C for 20 hours inactivated greater than 500 and less than 10,000 chimpanzee infectious doses (CID) of hepatitis B virus, while treatment at 98 degrees C for 30 minutes inactivated less than 500 CID. Both methods were successful in preventing infection with an undetermined amount of an indeterminate non-A, non-B hepatitis agent. The 60 degrees C, 20-hour treatment method rendered 5.25 logs of the putative acquired immune deficiency syndrome virus, human T-cell lymphotrophic virus III/lymphadenopathy virus, added to factor VIII or factor IX concentrates, undetectable. Heat-treated factor VIII and factor IX complex concentrates prepared by these methods were tested against corresponding untreated control lots. There was no significant difference in the plasma recovery or plasma half-life of the factor (p greater than 0.05). The treated concentrates were equivalent to the control concentrates with respect to vital signs, clinical laboratory studies, and adverse reactions. The heat-treated concentrates appeared bioequivalent to the untreated concentrates with the additional benefit of inactivation of potentially present infectious viruses.

Thermal inactivation of the acquired immunodeficiency syndrome virus, human T lymphotropic virus-III/lymphadenopathy-associated virus, with special reference to antihemophilic factor.

The virus that causes the acquired immunodeficiency syndrome (AIDS), human T lymphotropic virus/lymphadenopathy-associated virus (HTLV-III/LAV), was incubated at temperatures from 37 degrees to 60 degrees C and virus titer (ID-50) was determined over time by a microculture infectivity assay. The rate of thermal decay was consistent with first-order kinetics, and these data were used to construct a linear Arrhenius plot (r = 0.99), which was used to determine inactivation time as a function of temperature. In the liquid state, thermal decay was little affected by matrix (culture media, serum, or liquid Factor VIII). In the lyophilized state, the time required to reduce virus titer 10-fold (1 log) at 60 degrees C was 32 min compared with 24 s in the liquid state. HTLV-III/LAV in liquid antihemophilic Factor VIII or IX was lyophilized and heated according to commercial manufacturers' specifications. Infectious virus was undetectable with these regimens. Heat treatment should reduce or stop transmission of HTLV-III/LAV by commercial antihemophilic Factor VIII or IX.

Isolation, subunit structure, and proteolytic modification of bovine factor VIII.

A new method has been described for the isolation of factor VIII. The method results in a high yield of factor VIII that is homogeneous by several different criteria. The purified protein is very stable and is not dissociated in the presence of 1 M NaCl or 0.25 M CaCl2. The highly purified protein is readily activated and inactivated by various proteolytic enzymes, such as thrombin, plasmin, and trypsin. The molecular events that lead to the activation reaction, however, have not been established.