Viral safety of C1-inhibitor NF.

We studied the efficacy of virus reduction by three process steps (polyethylene glycol 4000 (PEG) precipitation, pasteurization, and 15nm virus filtration) in the manufacturing of C1-inhibitor NF. The potential prion removing capacity in this process was estimated based on data from the literature. Virus studies were performed using hepatitis A virus (HAV) and human immunodeficiency virus (HIV) as relevant viruses and bovine viral diarrhea virus (BVDV), canine parvovirus (CPV) and pseudorabies virus (PRV) as model viruses, respectively. In the PEG precipitation step, an average reduction in infectious titer of 4.5log(10) was obtained for all five viruses tested. Pasteurization resulted in reduction of infectious virus of >6log(10) for BVDV, HIV, and PRV; for HAV the reduction factor was limited to 2.8log(10) and for CPV it was zero. Virus filtration (15nm) reduced the infectious titer of all viruses by more than 4.5log(10). The overall virus reducing capacity was >16log(10) for the LE viruses. For the NLE viruses CPV and HAV, the overall virus reducing capacities were >8.7 and >10.5log(10), respectively. Based on literature and theoretical assumptions, the prion reducing capacity of the C1-inhibitor NF process was estimated to be >9log(10).

Differential sensitivities of pathogens in red cell concentrates to Tri-P(4)-photoinactivation.

BACKGROUND AND OBJECTIVES: Photodynamic treatment (PDT) with the cationic porphyrin, mono-phenyl-tri-(N-methyl-4-pyridyl)-porphyrin chloride [Tri-P(4)], has previously been shown to be effective at inactivating vesicle stomatitis virus (VSV) in red cell concentrates (RCC) with limited damage to red blood cells (RBC). The aim of this study was to determine the pathogen-inactivating capacity of PDT with Tri-P(4) for a broader range of pathogens and to establish the associated effect on in vitro RBC quality. MATERIALS AND METHODS: A series of viruses and bacteria was spiked into 60% RCC. Pathogen inactivation was determined after PDT with 25 microm Tri-P(4) and red light up to 360 kJ/m2. Human immunodeficiency virus (HIV)-infected cells were evaluated for cell death induction, and RCC were analysed for the induction of haemolysis and ATP content. RESULTS: For the lipid-enveloped viruses bovine viral diarrhoea virus, HIV and pseudorabies virus, and for the Gram positive bacterium, Staphylococcus aureus, and the Gram-negative bacteria, Pseudomonas aeruginosa and Yersinia enterolitica, inactivation of > or = 5 log10 was measured after 60 min of PDT with Tri-P(4). The required treatment time to achieve this level of inactivation was four times longer than required for VSV. For cell-associated HIV, only 1.7 log10 of inactivation was found, despite clear induction of cell death of HIV-infected cells. The non-enveloped virus, canine parvovirus, was completely resistant to the treatment. PDT of RCC with Tri-P(4) for 60 min, and subsequent storage in AS-3, resulted in 4% haemolysis after 35 days of storage. The ATP content of untreated and treated RBC declined with similar kinetics during storage. CONCLUSION: PDT of RCC with Tri-P(4) for 60 min inactivates a wide range of pathogens, but not cell-associated HIV and a non-enveloped virus, and compromises RBC quality. This reduces the suitability of PDT with Tri-P(4) for red cell sterilization. Therefore, further improvements in the treatment procedures to potentiate pathogen inactivation and to preserve RBC integrity will be required to generate an effective treatment for sterilizing RCC.

Viral safety of Nanogam, a new 15 nm-filtered liquid immunoglobulin product.

BACKGROUND AND OBJECTIVES: Producers of plasma derivatives continuously improve the viral safety of their products by, for example, introducing additional virus-reducing steps into the manufacturing process. Here we present virus-elimination studies undertaken for a number of steps employed in a new manufacturing process for liquid intravenous immunoglobulin (Nanogam) that comprises two specific virus-reducing steps: a 15-nm filtration step combined with pepsin treatment at pH 4.4 (pH 4.4/15NF); and solvent-detergent (SD) treatment. The manufacturing process also includes precipitation of Cohn fraction III and viral neutralization, which contribute to the total virus-reducing capacity of the manufacturing process. In addition, the mechanism and robustness of the virus-reducing steps were studied. MATERIALS AND METHODS: Selected process steps were studied with spiking experiments using a range of lipid enveloped (LE) and non-lipid-enveloped (NLE) viruses. The LE viruses used were bovine viral diarrhoea virus (BVDV), human immunodeficiency virus (HIV) and pseudorabies virus (PRV); the NLE viruses used were parvovirus B19 (B19), canine parvovirus (CPV) and encephalomyocarditis virus (EMC). After spiking, samples were collected and tested for residual infectivity, and the reduction factors were calculated. For B19, however, removal of B19 DNA was measured, not residual infectivity. To reveal the contribution of viral neutralization, bovine parvovirus (BPV) and hepatitis A virus (HAV) were used. RESULTS: For the pH 4.4/15NF step, complete reduction (> 6 log(10)) was demonstrated for all viruses, including B19, but not for CPV (> 3.4 but < or = 4.2 log(10)). Robustness studies of the pH 4.4/15NF step with CPV showed that pH was the dominant process parameter. SD treatment for 10 min resulted in complete inactivation (> 6 log(10)) of all LE viruses tested. Precipitation of Cohn fraction III resulted in the significant removal (3-4 log(10)) of both LE and NLE viruses. Virus-neutralization assays of final product revealed significant reduction (> or = 3 log(10)) of both BPV and HAV. CONCLUSIONS: The manufacturing process of Nanogam comprises two effective steps for the reduction of LE viruses and one for NLE viruses. In addition, the precipitation of Cohn fraction III and the presence of neutralizing antibodies contribute to the total virus-reducing capacity of Nanogam. The overall virus-reducing capacity was > 15 log(10) for LE viruses. For the NLE viruses B19, CPV and EMC, the overall virus-reducing capacities were > 10, > 7 and > 9 log(10), respectively. Including the contribution of immune neutralization, the overall virus-reducing capacity for B19 and HAV is estimated to be > 10 log(10).

Additive effects of dipyridamole and Trolox in protecting human red cells during photodynamic treatment.

BACKGROUND AND OBJECTIVES: Photodynamic treatment (PDT) of red blood cell (RBC) suspensions has been reported to result in virus inactivation, but also in deterioration of cell quality. Recently, we have demonstrated the potential usefulness of the reactive oxygen species scavenger dipyridamole in selectively protecting RBCs against the harmful side-effects of PDT. Unfortunately, dipyridamole-conferred protection against long-term photohaemolysis was incomplete. In the present study, dipyridamole was applied in combination with Trolox (a hydrophilic vitamin E analogue) in order to augment RBC protection. MATERIALS AND METHODS: Leucodepleted RBC suspensions (30% haematocrit) were treated with 1,9-dimethylmethylene blue (DMMB) and red light, and the effect of inclusion of dipyridamole and Trolox was assessed on potassium leakage as well as on short-term and long-term photohaemolysis. Possible interference of the scavenger cocktail with virus inactivation was examined using extracellular pseudorabies virus (PRV). RESULTS: Treatment of RBC with DMMB and red light resulted in enhanced potassium leakage and both short- and long-term haemolysis. Dipyridamole and Trolox showed additive protective effects against induction of potassium leakage and photohaemolysis, suggesting different protection mechanisms for the two scavengers. Combined inclusion of dipyridamole and Trolox did not interfere with efficacy of PRV inactivation. CONCLUSIONS: Combined inclusion of dipyridamole and Trolox results in substantially improved selectivity of photodynamic treatment of RBC suspensions.

The virucidal spectrum of a high concentration alcohol mixture.

The virucidal spectrum of a high concentration alcohol mixture (80% ethanol and 5% isopropanol) was determined for a broad series of lipid-enveloped (LE) and non-lipid-enveloped (NLE) viruses covering all relevant blood-borne viruses. LE viruses were represented by human immunodeficiency virus (HIV), bovine viral diarrhoea virus (BVDV), a specific model virus for hepatitis C virus (HCV), pseudorabies virus (PRV), and vaccinia virus. For the NLE viruses hepatitis A virus, canine parvovirus (a model for human parvovirus B19), and reovirus type 3 (Reo-3) were used. PRV, vaccinia, and Reo-3 served as general model viruses. The alcohol mixture was spiked with 5% (v/v) virus, mixed and tested for residual virus after 5 min treatment. Complete clearance (reduction by a factor of >10(6)) was observed for LE viruses, whereas incomplete to insignificant clearance (ranging from no reduction up to a maximum factor of 10(4)) was found for NLE viruses. In a second series of spiking experiments using the LE viruses BVDV, HIV, and PRV, complete clearance (reduction by a factor of >10(6)) was found after 20 s treatment. These data strongly suggest that treatment with a high concentration alcohol mixture has a high virucidal potential in particular for the blood-borne LE-viruses HIV, hepatitis B virus, and HCV. Such mixtures are well suited for rapid and frequent disinfection in dental practice being non-hazardous and non-toxic.

The band III ligand dipyridamole protects human RBCs during photodynamic treatment while extracellular virus inactivation is not affected.

BACKGROUND: Recently, the potential usefulness of dipyridamole (DIP) in protecting RBCs against the harmful side effects of photodynamic sterilization was demonstrated. In the present study, the use of DIP for selective protection of RBCs was investigated under conditions more relevant for blood bank practice. STUDY DESIGN AND METHODS: WBC-reduced RBC suspensions (30% Hct) were treated with 1,9-dimethylmethylene blue and red light, and the influence of the inclusion of DIP on photohemolysis was assessed as a function of sensitizer concentration, light dose, and storage time. Furthermore, the possible interference of DIP with inactivation of extracellular virus by use of a panel of different viruses (HIV-1, pseudorabies virus [PRV], bovine viral diarrhea virus [BVDV], VSV, encephalomyocarditis, and canine parvovirus) was investigated. RESULTS: In WBC-reduced RBC suspensions (30% Hct), DIP exerted a clear protective effect against photohemolysis. Part of this protection was achieved with concentrations near the dissociation constant for band III binding. Importantly, efficiency of inactivation of extracellular HIV-1, PRV, BVDV, and VSV was not significantly impaired by the inclusion of DIP. Phototreatment conditions, resulting in a 4 to 5 log inactivation of extracellular HIV-1 and PRV, resulted in a high level of hemolysis after 28 days of storage. This long-term hemolysis could be decreased, but not completely prevented, by the inclusion of DIP. CONCLUSION: Photohemolysis in RBC concentrates can be reduced substantially by the application of DIP, while the efficacy of inactivation of HIV-1 and other viruses remains unchanged.