Virus validation studies

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. 

A major issue in performing virus validation studies is determining which viruses should be used. The Committee for Proprietary Medicinal Products (CPMP) has issued guidelines on the selection of viruses to evaluate in validation studies. Processes must be validated for their capacity to inactivate/remove relevant viruses, or viruses that are known to contaminate plasma or other materials in the production process. If relevant viruses cannot be easily propagated in cell culture or assayed, then validation studies should include specific model viruses with characteristics similar to relevant viruses. If relevant viruses do not represent viruses with... 

The data also demonstrated that the subtle differences in viruses should not be underestimated. The different HAV strains/clones and the method of virus preparation had a significant impact on virus clearance. Virus stocks used in virus validation studies are produced in cell culture and the behavior of tissue culture derived viruses may be different from that of native viruses. Laboratory adapted strains of viruses may also have impre-dicted properties, such as association with lipids, which... 

Virus validation studies assess the virus clearance capacity of a process and the evaluation and interpretation of virus clearance data from terminal inactivation and upstream process steps are similar. Limitations in the design and execution of virus validation studies may lead to an incorrect estimate of the ability of a process to inactivate/remove virus infectivity. The three terminal inactivation treatments discussed here illustrate the importance of rigorously controlling virus validation studies. 

Committee for Proprietary Medicinal Products (CPMP). Note for Guidance on Virus Validation Studies, The design, contribution and interpretation of studies validating the inactivation and removal of viruses. CPMP/ BWP/268/95, 1996. 

CPMP/BWP/268/95 Note for Guidance on Virus Validation Studies The Design, Contribution and Interpretation of Studies validating the Inactivation and Removal of Viruses (CPMP adopted February 1996). 

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. 

Table 6 Some Variables in Virus Clearance Validation Studies... 

For such validation studies, one of the critical factors is the choice of viruses. This will unavoidably be dependent on the starting raw material in the case of monoclonal antibody purification, which is frequently done from rodent cell line cultures, retroviruses are of particular concern. 

Arindam Bose (Pfizer Central Research) further discussed the ICH documents and presented a rationale for the recommended combination of test procedures and process clearance validations required to demonstrate that marketed biopharmaceuticals are free of adventitious agents. He showed that testing of Pre-Seed Stock (PSS), the Master Cell Bank (MCB), and the Working Cell Bank (WCB) is required to demonstrate that they are free from contamination by mycoplasma, bacteria, molds, and yeasts. In addition, viral clearance validation studies must be performed on scaled down versions of each chromatographic step and the viral inactivation/removal step employed in the product purification scheme. Finally, clearance studies must be conducted with a panel of relevant and model viruses (typically three to four) to establish that the purification scheme will indeed purge any viruses that may be inadvertently introduced during processing. 

In summary, the large number and complexity of the variables to control when implementing a terminal freeze dry/dry heat treatment makes validation very difficult and increases the probability for error. Since the reliability of the results from virus clearance studies is dependent on the appropriateness of the models used in the studies, it is essential that the freeze dry/dry heated material and the experimental conditions used at small scale be representative of production scale. 

Federal Health Agency and Paul Ehrlich Institute Federal Agency for Sera and Vaccines. Announcement on the Marketing Authorization of Medicinal Products Requirements of Validation Studies for Demonstrating the Virus Safety of Medicinal Products Derived from Human Blood or Plasma. Federal Gazette No. 84, 4 May 1994. 

The models can be scaled to various sizes to fit the needs of the experiment. For example, a very small-scale nanofiltration system, such as a Planova P-15 hollow-fiber cartridge with O.OOl-m surface area (Asahi Kasei Corporation, Japan), can be used to study virus retention capabilities of a virus reduction step in a biological manufacturing process, whereas a scaled-up version of the same system with a surface area of 0.01 m provides an excellent way to study the nanofiltration process variables. In a nanofiltration validation study, a feed sample is typically spiked with a known quantity of a model virus. The mixture is filtered under the expected process... 

Since most immortalized cell lines have been shown to express endogenous retro-virus-like particles that may or may not be replication-competent and infectious, and other virus classes may also be present in mammalian cells, the risk for a virus contamination of the end product is inherent. In addition, adventitious vimses may come into contact with the product during processing. In any case, cell culture processes must demonstrate the robust and rehable abihty to eliminate viruses in a risk-based approach [162] (see Part IV, Chapter 1). Vims validation studies employ model vimses that are relevant in representing known risks from the sources involved [163, 164]. 

The capacity of a pnrification process to clear viruses is demonstrated at a representative small scale nsing model viruses. The most common model viruses used in this validation study are xenotropic murine leukemia virus (x-MuLV), mouse minute virus (MMV), and reovirus (Reo). The viral clearance capacity of the chromatographic steps, inactivation steps, and the viral filnation step is demonstrated by spiking a known amount of a model virus into the load of each of these unit operations and calculating the efhciency of removal by measuring the remaining viral titer in the product containing fractions. 

I, virus validation must be conducted. Typical viruses studied are the murine leukemia virus as a model virus for retrovirus-like particles produced by murine cell expression systems (if applicable to the process) and parvovirus [50]. Before phase... 

Scale-down studies have been used for a wide variety of process validation studies, including resin lifetimes, in-process stream hold times, buffer stability, virus clearance, harvest criteria, filter extractables, resin leachables, and cell age at harvest [14, 91, 92]. The ease of scale-down differs depending on step and should be considered in selecting those steps to be validated [5]. In fact, certain validation issues can be addressed only via small-scale models (e.g., virus clearance evaluation, nucleic acid and other impurities/additives removal, cleaning and storage procedure evaluation, and column lifetime estimation) because their use increases worker safety, reduces costs, and permits use of higher titer samples for improved... 

To determine if the high in vitro potents of the anti-HIV compound 30 translates into antiviral efficiency in vivo, Datema et al. investigated the inhibition of HIV-1 production and of depletion of human T cells in HIV-1-infected SCID-hu Thy/Liv mice [37]. Steady levels of 100 ng of 30 or higher per mL in plasma resulted in significant inhibition of HIV p24 protein formation. Daily injection of 30 caused a dose-dependent decrease in viral p24 production, and this inhibition could be potentiated by coadministration of AZT (or DDI). This study suggested that 30 alone or in combination with the licensed anti-HIV agents AZT and DDI may decrease the virus load in HIV-infected patients and, by extension, that the infectious cell entry step is a valid target for antiviral chemotherapy of HIV disease. 

Filters are used for clarification, removal of small molecules, exchange of buffers, and concentration of product, as well as sterilization and virus removal. A recent review of validation of filtration describes the critical validation issues [29], Filter compatibility is tested with process conditions to avoid nonspecific binding of product to the filter or addition of extractables to the process stream. Extractables are defined and limits established based on final product safety studies. Special considerations apply for sterilizing filters and those that are designed for virus removal. These filters are single use, however, which simplifies the validation effort. 

Validation of viral clearance is a major concern for products derived from mammalian cell culture and transgenic animals, as well as for viral vectors used for gene therapies. As we learn more and more about potential risks from newly found viruses, the requirements for validation increase. The increased concerns may be reflected in the number and types of viruses that are used for viral clearance studies. Both relevant and model viruses are used. A recent review of validation of the purification process for viral clearance evaluation provides further information on selection of viruses and performance of the studies [36],... 

Several documents describe the requirements for viral clearance studies. The ICH guidance on viral safety evaluation provides information on the design of viral clearance studies and their interpretation [37], Unlike most other aspects of process validation, viral clearance cannot be performed at full scale. There are several reasons for this. Direct testing methods may not detect low concentrations of virus, which requires that viruses be spiked into the feedstream. Assays may detect only known viruses, and they may also fail to detect variants. Worker safety is another issue that necessitates the need to perform the validation at a small scale. Scaling down is addressed in the ICH guidelines and in the literature [38,39]. 

The specificity of PCR should make validation efforts for the clearance of viruses, DNA, and host cell proteins more efficient by combining studies and increasing the speed of the assays. In the case of DNA, the assay sensitivity may enable validation to be performed at full scale and eliminate the need for more costly and less accurate small-scale spiking studies. 

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