Peter L. Roberts

Virus inactivation by high intensity broad spectrum pulsed light.

The inactivation of a range of enveloped and non-enveloped viruses by treatment with high intensity broad spectrum pulsed light (PureBright) has been investigated. In phosphate buffered saline, a dose of 1.0 J/cm2 was sufficient to effectively inactivate, i.e. >4.8->7.2 log of all the viruses tested, i.e. Sindbis, HSV-1, vaccinia, polio-1, EMC, HAV, CPV, BPV and SV40. However, in the presence of protein, i.e. 5% v/v foetal-calf serum (0.2% w/v protein), virus inactivation was less effective. At a dose of 2.0 J/cm2, virus inactivation was 5.0->6.4 log, however, HSV-1 (3.8 log), BPV (2.4 log) and SV40 (2.9 log) were all relatively resistant. This virus inactivation procedure may have application for increasing the safety of therapeutic biological products.

Virus inactivation by solvent/detergent treatment using Triton X-100 in a high purity factor VIII

Virus inactivation by solvent/detergent treatment using 0.3% tri-n-butyl phosphate and 1% Triton X-100 in the high purity factor VIII concentrate Replenate has been investigated. A wide range of model enveloped viruses were confirmed to be inactivated by >4 to >6 log after 30 min at 22 degrees C under standard conditions. Using Sindbis as a representative enveloped virus, the effect of various parameters on the inactivation process was tested. Virus inactivation was confirmed to be effective in different batches of product and was not influenced by changing the process conditions with regard to protein and salt concentration or pH. Virus inactivation was effective even at a temperature as low as 4-5 degrees C. Although solvent/detergent concentration was the most critical parameter, a concentration as low as 0.15% TnBP/0.5% Triton X-100 was still completely effective. At a lower concentration an extended incubation period was required. These studies demonstrate the robustness of this solvent/detergent procedure based on Triton X-100 and allow suitable process limits to be set for this manufacturing step.

Removal and Inactivation of Enveloped and Non-Enveloped Viruses during the Purification of a High-Purity Factor IX by Metal Chelate Affinity Chromatography

Virus reduction during the copper chelate affinity chromatography stage used during the purification of a new high‐purity factor IX (BPLs 9MC) has been investigated. Virus reduction for the enveloped virus Sindbis was 6.5 log, a value which included approximately 2 log of inactivation due to the use of an acidic wash buffer (pH 4.4) during chromatography. In the case of the non‐enveloped hepatitis‐A‐like poliovirus, which is acid‐resistant, the virus reduction value was 4.0 log and was exclusively due to physical virus removal during the chromatographic process.

Virological safety of the purified factor IX concentrate, Replenine

Virus safety is an important consideration with any coagulation factor concentrate. The viruses of particular concern include hepatitis B, Delta virus, hepatitis C, the human immunodeficiency virus (HIV) and the human parvovirus B19 [I]. In addition, there have been some reports of hepatitis A transmission [l], but these have been associated primarily with a specific type of factor VIII product manufactured in Europe. The intermediate purity factor IX concentrate (9A), containing factors 11, IX and X, uses severe dry heat-treatment at 80°C for 72 h for virus inactivation. Because of concerns over the thrombogenic potential of factor IX concentrates of this type (PCCs), a new high-purity single factor IX product (Replenine) has been developed [2]. For this product, a relatively gentle procedure, i.e. solvent/detergent, was included as a major virus-reduction step. This approach has the advantage that product activity losses are very low and that neoantigens are not formed as might occur with a more severe method such as heat treatment. In this high-purity factor IX, a number of strategies are used to ensure the virus safety of the product. These include the use of non-remunerated UK donors, virus screening, virusreduction steps, patient monitoring following transfusion of concentrates and vaccination of recipients against hepatitis A and B. Screening for viruses is carried out either by antigen detection in the case of hepatitis B (surface antigen) or indirectly by the detection of antibody in the case of HIV 1 + 2 and hepatitis C. The screening process is performed on each donation, and thus the sensitivity is maximized. In addition, further screening is carried out at the plasma pool stage and on the final product. There are limitations associated with this type of approach, including the volume of sample that can be tested, the inherent sensitivity of the test, and the window period during which the relevant antibody is absent but the virus is present. Virus reduction during the manufacturing process occurs at two principal stages. These are the inactivation of enveloped viruses by solvenddetergent treatment and the removaUinactivation of viruses during metal chelate affinity chromatography. In order to investigate these steps, laboratory studies using a range of model and relevant viruses have been carried out. Experimental approach

Effect of manufacturing process parameters on virus inactivation by solvent–detergent treatment in a high‐purity factor IX concentrate

Background and Objectives  Treatment with solvent–detergent is widely used for ensuring the virus safety of plasma products. Laboratory studies have shown this procedure to be effective for inactivating enveloped viruses under manufacturing conditions. In the present study, the effect of different manufacturing process parameters on virus inactivation by treatment with polysorbate 80 and tri‐n‐butyl phosphate were investigated for a high‐purity factor IX concentrate in order to evaluate the robustness of this step.

Virus inactivation in a factor VIII/VWF concentrate treated using a solvent/detergent procedure based on polysorbate 20.

Treatment with solvent/detergent is a widely used method for ensuring the virus safety of plasma products. In the present study, virus inactivation by a novel solvent/detergent combination, i.e. TnBP (tri-n-butyl phosphate) and polysorbate 20 during the manufacture of the factor VIII/VWF concentrate Optivate has been investigated. The inactivation of most enveloped viruses was rapid, i.e. > 5 log in 2 min, although the inactivation of vaccinia virus was slower, i.e. 4 log in 1h. Virus inactivation was effective over a wide range of conditions, i.e. solvent/detergent concentration, protein concentration and temperature, irrespective of whether tested individually or in combination. This confirms the effectiveness and robustness of this alternative version of the solvent/detergent procedure, and allows appropriate control limits to be set for this manufacturing step. Polysorbate 20 provides an alternative to the non-ionic detergents currently in use with the solvent/detergent procedure.

Efficient removal of viruses by a novel polyvinylidene fluoride membrane filter

Virus removal by a novel filter (Ultipor VF DV50), comprising three layers of PVDF membrane, has been evaluated by infectivity studies using a range of viruses and conditions. The filter was able to remove at least 6 log of various viruses, i.e. Sindbis and Semliki Forest (40-70 nm), herpes simplex (120-200 nm) and vaccinia (200 x 350 nm), from cell-culture medium or phosphate buffered saline pH 6.8 containing 0.5% albumin. However, the removal of polio virus (25-30 nm) under these conditions was only limited, i.e. about 1 log. This filter is thus effective for removing viruses of about 50 nm or larger. Proteins as large as immunoglobulins (MW 160,000), were able to pass through the filter with recoveries of at least 85%. Due to its ability to remove viruses of medium to large size, this filter shows potential for increasing the safety of biological products where viruses such as hepatitis B, C, herpes and retroviruses are of concern.

Effect of manufacturing process parameters on virus inactivation by dry heat treatment at 80 °C in factor VIII

Background and Objectives  Dry heat treatment at 80 °C for 72 h is used as a virus inactivation step for some coagulation factor concentrates such as Bio Products Laboratorys (BPL) factor VIII 8Y. In the current study, the effect of this process has been tested on a range of viruses. In addition the effect of various manufacturing process parameters on virus inactivation has been investigated.

Inactivation of parvovirus B19 and model viruses in factor VIII by dry heat treatment at 80°C

1648 TRANSFUSION Volume 46, September 2006 Atlanta, Georgia e-mail:bis3@cdc.gov Indira Hewlett, PhD Leslyn Aaron, BS Laboratory of Molecular Virology Division of Emerging and Transfusion Transmitted Diseases OBRR/CBER/FDA Rockville, Maryland Nathan D. Wolfe, ScD Donald S. Burke, MD Departments of Epidemiology International Health Bloomberg School of Public Health Johns Hopkins University Baltimore, Maryland Walid Heneine, PhD Laboratory Branch Division of HIV/AIDS Prevention National Center for HIV, Hepatitis, STD, and TB Prevention Centers for Disease Control and Prevention Atlanta, Georgia

Virus removal from factor IX by filtration: validation of the integrity test and effect of manufacturing process conditions.

Virus removal from a high purity factor IX, Replenine-VF, by filtration using a Planova 15N filter has been investigated. A wide range of relevant and model enveloped and non-enveloped viruses, of various sizes, were effectively removed by this procedure. Virus removal was confirmed to be effective when different batches of filter were challenged with poliovirus-1. It was confirmed that intentionally modified filters that failed the leakage test had completely lost the ability to remove virus, thus confirming that this test demonstrates gross filter failure. In the case of the more sensitive integrity test based on gold particle removal, it was found that a pre-wash step was not essential. Planova filters that had been modified by sodium hydroxide treatment to make them more permeable, and filters manufactured with varying pore-sizes over the range of 15-35 nm, were tested. The integrity test value that resulted in the removal of >4 log(10) of poliovirus-1 from the product correlated with that recommended by the filter manufacturer. Virus removal from the product was not influenced by filter load mass, flow-rate or pressure. These studies confirm the robustness of this filtration procedure and allow suitable process limits to be set for this manufacturing step.