Virus reduction factors

The virus reduction factor of an individual purification or removal—inactivation step is defined as the log10 of the ratio of the virus load in the pre-purification material divided by the virus load in the post-purification material. A clearance factor for each stage can be calculated and the overall clearance capacity of the production process assessed. Total virus reduction is calculated as the sum of individual log reduction factors. Individual manufacturing steps must possess fundamentally different mechanisms of virus removal or inactivation in order for values to be considered cumulative. Additionally, because viruses vary greatly with regard to inactivation or removal profiles, only data for the same model virus can be cumulative. 

Contaminant-clearance validation studies are of special signibcance in biopharmaceutical manufacture. As discussed in Section 7.6.4, downstream processing must be capable of removing contaminants such as viruses, DNA and endotoxin from the product steam. Contaminant-clearance validation studies normally entail spiking the raw material (from which the product is to be purihed) with a known level of the chosen contaminant and subjecting the contaminated material to the complete downstream processing protocol. This allows determination of the level of clearance of the contaminant achieved after each purihcation step, and the contaminant reduction factor for the overall process. 

Plasma-derived therapeutic proteins are parenteral biologies that are purified on an industrial scale. All biologies derived from human sources, such as plasma, carry the risk of viral contamination. Thus, in order to market a medicinal product derived from human plasma, manufacturers must assure the absence of specific viral contamination. Virus validation studies are performed to evaluate the capacity of a manufacturing process to remove viral contaminants. Virus clearance across three different terminal inactivation steps, low pH incubation of immunoglobulins (IgG), pasteurization of albumin, and freeze dry/dry heat treatment of plasma-derived products (Factor VIII and Protein G), is discussed in this article. The data show that, like all other upstream virus reduction steps, the methods used for terminal inactivation are process and product dependent, and that the reduction factors for an individual step may be overestimated or underestimated due to inherent limitations or inadequate designs of viral validation studies. 

The overall virus reduction capacity of a manufacturing process is the sum of the individual reduction factors, and should be greater than the potential virus load in the starting material. At least one step in the process should clear significant levels of infectious virus so that the overall clearance is not made up of individual small, and possibly negligible, reductions. 

The viral clearance reduction factor, the common logarithm of the ratio between the total virus loads before and after clearance, is established for viruses known to contaminate the production process [50]. Individual step clearances are combined to obtain the total clearance reduction factor. This reduction factor is used in combination with an assessment of step robustness to classify the step as effective (>4 reduction factor and unaffected by small changes in process variables), moderately effective (4 > reduction factor > 1), or ineffective (<1 reduction factor) with respect to virus clearance [50]. Clearance factors are usually multiplied if the mechanism is different for two separate steps and sometimes are added if the mechanism is same [51]. In other cases, if two independent steps have similar mechanisms of clearance, only one step is included in the summation because virus particles removed via that mechanism would only be expected to be removed in the first step [3, 5]. A total clearance of 12-15 logs is desired for lipid-enveloped viruses and fewer logs for nonenveloped viruses (e.g., polio) [30]. 

A low-level sensitivity is required for viral test methods to maximize reduction factors [5]. However, the ability to detect low virus concentrations is limited by... 

Interestingly, the persistence of viruses such as polio has been shown to be dependent on the vegetable type. When introduced onto lettuce or cabbage, a 1 log reduction in polio virus was observed over 8 days. In contrast, viruses introduced onto green onions remained stable for over 14 days (Kurdziel et al., 2001). The underlying factors associated with the persistence of enteric viruses on fresh produce remain to be elucidated. 

E Role in therapy Antihemophilic factor is indicated for the treatment of bleeding episodes or perioperative treatment in patients with hemophilia A. Prophylactic use has also been advocated for the prevention and/or reduction of bleeding episodes. The largest issue in treatment with antihemophilic factor is the choice of formulations because of the relative risk of viral transmission. Recombinant factor VIII has the lowest risk of transmission of blood-borne viruses, but its use may be limited due to cost and availability. 

The use of animals for the preclinical evaluation of blood derivatives primarily encompassed evaluation for activity [4], and later for viral contamination, but little from the perspective of actual toxicological endpoints. Many of the hemophiliacs who were treated with early versions of antihemophilic factor (AHF) and Factor IX became infected with hepatitis [5], This provided the major reason for developing viral inactivation methods for AHF concentrates. The hepatitis agents were referred to as non-A, non-B hepatitis (NANB). The preclinical demonstration that the active virus had been inactivated required the use of chimpanzees, which were injected with the AHF concentrate. During the early 1980s plasma fractionators used chimpanzee studies to demonstrate the effectiveness for the reduction of HBV and NANB hepatitis infectivity [6], Ultimately, previously untreated patients were evaluated in clinical trials that demonstrated the utility of a number of viral reduction methods for what was by then known as hepatitis C [7],... 

Because, with free chlorine, a 1-log Giardia inactivation provides greater than a 4-log virus inactivation, inactivation of Giardia is the controlling factor for determining overall reductions. 

Several different plasma-derived factor VEI products are avaUable (see Table 100 ). These products are derived from the plasma of thousands of donors, and therefore potentially can transmit infection. Donor screening, testing plasma pools for evidence of infection, viral reduction through purification steps, and viral inactivation procedures (e.g., dry heat, pasteurization, and solvent detergent treatment) have all resulted in a safer product. No cases of HIV transmission from factor concentrates have been reported since 1986. However, there have been isolated reports of hepatitis C infection with the use of plasma-derived products. Additionally, there have been outbreaks of hepatitis A viral infections associated with plasma-derived products, likely because solvent detergent treatment does not inactivate this nonenveloped virus. Parvovirus has also been reported to be present in both plasma-derived and recombinant factor VIII products. " Finally, there remains concern about the possibility for infection with as yet unidentified viruses that currently used methods would not inactivate. 

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