Virus clearance validation

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

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. 

Darling, A. 2002. Validation of biopharmaceutical purification processes for virus clearance evaluation. Molecular Biotechnology 21, 57-83. 

Dabbah, R. Grady, L. (1998). Pharmacopoeial harmonization in biotechnology. Curr. Opin. Biotechnol. 9, 307-311. Darling, A. (2002). Validation of biopharmaceutical purification processes for virus clearance evaluation. Mol. Biotechnol. 21(1), 57-83. 

Prior to phase I clinical trials, process steps and assays that relate to safety should be validated. For example, sterility assays and sterilization processes must be validated. Cell lines should be qualified prior to any clinical trials, including testing for adventitious agents and identifying and quantifying indigenous virus. Virus clearance steps should be validated, and removal of any potentially toxic or otherwise harmful agents should be validated [41,42],... 

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. 

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 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... 

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. 

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. 

Validation of the virus clearance capability of the production process. 

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... 

Validation of viras clearance requires determining the clearance for a number of different unit operations and then summing the individual clearance factors to obtain the overall clearance by the purification train. If the overall clearance is less than the required clearance, additional unit operations must be added to the purification train simply to validate adequate virus clearance. However, addition of new unit operations to validate vims clearance increases the manufacturing cost. Consequently, there is a tremendous need to validate vims clearance in existing unit operations. 

Tangential-flow filtration could also be used to validate virus clearance. Today most virus clearance filters are operated in normal flow mode. However, when the size of the model virus particle for which clearance is being validated, and the desired product are within an order of magnimde of each other, tangential-flow filtration may be the preferred mode of operation. 

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. 

The risk associated with the presence of viruses justified clearance studies that are one of the important places of safety documentation for final pure antibodies. These studies are referred to as viral validation and are based on clearance effect of extraction-purification steps and on virus removal-inactivation steps. A comprehensive rational approach to guaranteeing a minimum risk has been reported by Berthold et al.235... 

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. 

Steps to be Validated for Viral Clearance. Process validation for viral clearance is conducted only on robust steps that can (1) be scaled down accurately and (2) reproducibly and effectively remove and/or inactivate a wide variety of potential viral contaminants under a wide variety of process conditions [5,41]. The number of steps selected for validation depends on estimated viral clearance effectiveness based on historical data and target clearance values [5]. The FDA demands at least two different steps for virus reduction to guarantee safety and efficacy [7]. Potentially only two steps are required for antibody processes that use serum-free medium, but additional steps might be required if viral contamination risk is increased by using serum-containing medium [7]. Due to the use of live viruses to perform clearance studies, this work usually is outsourced to reduce cross-contamination issues [14]. 

At the preclinical product phase, critical and noncritical classification of process input parameters should be initiated [32]. Critical components of facility subsystem validation need to be essentially complete before phase I product manufacture [15]. For phase I, it is necessary to validate aspects of the process related to product safety (e.g., sterility, mycoplasma, viral clearance, impurity removal, and stability) [14]. Abbreviated viral clearance studies for model viruses/retroviruses and impurity clearance studies for host cell DNA often are acceptable, resulting in fewer downstream steps validated at this product stage [3, 5]. If viral clearance results are available in sufficient time, the results can be applied to developing the phase I process steps. All assays do not have to be validated at this stage, but some (especially product-specific ones) should be at least qualified [14]. 

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