Proceedings of the 2009 Viral Clearance Symposium.

The 2009 Viral Clearance Symposium (Indianapolis, IN, USA) was held to interactively discuss methods for virus removal and inactivation during biopharmaceutical manufacture. Its origin was the result of worldwide regulatory and industry recognition that challenges, gaps, and opportunities for improvement exist, which if formally addressed could benefit the field as a whole. The symposium began with presentations by the FDA (USA) and the Paul Ehrlich Institute (PEI, Germany), which highlighted viral clearance study information reported in regulatory submissions. In these two presentations, and a subsequent series of brief industry presentations covering various unit operations, it was made clear that many unit operations are quite effective in clearing viruses. This was particularly true of low pH inactivation, anion exchange chromatography, and virus filtration. Moreover, the follow-up discussions at the end of each session, and the wrap-up at the end of the symposium, aimed to synthesize the regulatory data mining knowledge base with the industry-generated data. The symposium also revealed a number of unknowns in the field which were defined and prioritized, and served as potential action items for future experimental studies.

Parvovirus B19 DNA in plasma pools and plasma derivatives.

BACKGROUND AND OBJECTIVES: Human parvovirus B19 (B19) has been transmitted by various plasma-derived medicinal products. The aim of this study was to determine the frequency and the level of B19 DNA contamination in plasma pools destined for fractionation and in a broad range of plasma derivatives. In addition, removal of B19 DNA by the manufacturing process was investigated in cases where corresponding samples from plasma pool and product were available. MATERIALS AND METHODS: Plasma pool samples and blood products were tested for B19 DNA by nested polymerase chain reaction (PCR), and the viral DNA content was determined by TaqMan quantitative PCR. RESULTS: Two-hundred and twenty two of 372 plasma pools for fractionation contained B19 DNA at concentrations of 10(2)-10(8) genome equivalents/ml (geq/ml). While approximately 65% of the DNA-positive plasma pools were only moderately contaminated (< 10(5) geq/ml), 35% contained > 10(6) geq/ml. High frequencies of contamination were detected in Factor VIII (79 of 91), prothrombin complex concentrates (38 of 43) and Factor IX (41 of 62), where the concentration of B19 DNA ranged between 102 and 107 geq/ml. A lower level of B19 DNA contamination was found in antithrombin III (five of 26 samples), in anti-D immunoglobulins (three of 37 samples) and in albumin (four of 51 samples), with levels ranging between 10(2) and 10(3) geq/ml. Furthermore, investigation of plasma pools for solvent/detergent plasma (S/D plasma), from two manufacturers, revealed B19 DNA in 15 of 66 batches at concentrations of 10(2)-10(8) geq/ml. Similar concentrations were detected in the corresponding final S/D plasma products. Anti-B19 immunoglobulin G (IgG) was found in plasma pools and S/D plasma at concentrations of approximately 40 IU/ml. CONCLUSION: Although positive PCR results do not necessarily reflect infectivity, these data show that B19 is a common contaminant in plasma pools and in plasma-derived medicinal products. Considering the resistance of animal parvoviruses to inactivation by heat and chemical agents, and the absence of specific information for B19, the risk of B19 transmission by plasma products should be considered. Physicians should be aware of this problem when treating patients of B19-related risk groups. The plasma fractionation industry should continue their efforts to avoid B19 contamination of plasma derivatives and develop methods which are effective in removing/inactivating parvovirus B19.

Safety issues for plasma derivatives and benefit from NAT testing.

Manufacturing processes for plasma derivatives are in general highly effective for removal or inactivation of enveloped viruses and the products are safe with regard to the clinically important viruses HIV, HCV and HBV. They are not so effective for the elimination for non-enveloped viruses, especially Parvovirus B19 (B19). A certain risk remains of B19 contamination for some plasma derivatives that is caused, firstly, by the occurrence of highly contaminated donations (up to 10(14)genomes/ml) and secondly, by the extreme heat resistance and small size of B19 which makes it difficult to remove or inactivate. NAT is a beneficial tool for detection of virus contamination. It is routinely used for the detection of HCV-RNA in plasma pools, thereby preventing the processing of HCV-RNA positive material. NAT assays may also be valuable for testing the removal of viruses during manufacturing. This may be especially important if a virus cannot be tested by infectivity assays.

Prevalence of hepatitis C virus in plasma pools and the effectiveness of cold ethanol fractionation.

Screening blood donations for antibodies against hepatitis C virus (HCV) greatly reduces the risk of transmitting HCV by transfusions. However, despite such screening programs, plasma pools still contain a high percentage of HCV ribonucleic acid as determined by polymerase chain reaction. This result would not be alarming if the procedures for producing blood products included steps to inactivate or remove HCV. Although this appeared to be the case for all blood products, such as coagulation factors and most immunoglobulins, which are subjected to an inactivation step, the effectiveness of the cold ethanol fractionation process still needed to be determined. In validation experiments using bovine viral diarrhea virus as a model virus for HCV, we demonstrated that the Cohn-Oncley cold ethanol fractionation process neither inactivated nor removed this virus sufficiently. Our observations may help to explain how HCV was transmitted to a number of recipients of intravenous immunoglobulin.

The influence of pH and ionic strength on the single radial immunodiffusion test in qualitative assay of influenza virus haemagglutinin.

A significant dependence between different ionic strength and pH value of virus suspension on one hand and the haemagglutinin (HA) content as determined by single-radial-immunodiffusion (SRD) test on the other hand was observed after cleavage of influenza virus recombinant NIB-6 (H1N1) with sodium lauroyl sarcosinate (SLS). In contrast, no such relationship was found when the HA Of NIB-4 (H3N2) recombinant strain was determined.