Viral clearance studies

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]. 

During phase III any effects of scale changes should be validated and multiple lots placed on stability test. Extensive viral clearance studies should be... 

II, two to four additional viruses are selected that depend on the host cell and media origin [50]. It is advised to solicit feedback on virus selection from the relevant regulatory agencies depending on where the trials are to be conducted before conducting viral clearance studies for product destined for initial clinical trials [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]. 

For purification, scale-up considerations are important even in the earliest phases of development. It is important to avoid the use of purification techniques of limited scale-up potential even for early clinical production because thorough justification of process changes and demonstration of biochemical comparability are necessary prior to product licensure. For successful scale-up, it is important to understand the critical parameters affecting the performance of each purification step at each scale. Conversely, it is important to verify that the scaled-down process is an accurate representation of the scaled-up process, so that process validation studies, such as viral clearance and column lifetime studies, can be performed at the laboratory scale. 

Viral detection assays based on infectivity suffer from significant variability, which necessitates the use of statistical evaluation. Polymerase chain reaction-based assays are currently being developed and validated for viral clearance. With PCR assays, there is a potential to distinguish between inactivation and physical removal, perform mass balance studies, evaluate more than one vims at a time for a given process step, reduce the time for completing clearance studies, and accurately quantitate the amount of vims bound to such surfaces as chromatography resins. Table 5 compares the assay precision between an infectivity assay and a quantitative PCR assay. 

A few other issues related to process validation are under discussion. One is resin lifetime. Some firms are proposing concurrent validation rather than generating prospective laboratory scale for the entire lifetime. The concurrent approach would probably require more in-process testing, but the data generated may be more reliable since they are obtained at manufacturing scale. Clearly, eliminating the small-scale studies at this time for steps in which viral clearance is claimed will be quite difficult if not impossible. 

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... 

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 influenza virus host resistance model has been characterized in mice and rats, and has been widely used to evaluate the potential immunotoxicity of therapeutics. Influenza virus is used as the infectious challenge agent and administered intranasally in a 28-day repeat-dose study. Mice or rats are dosed for 7 days, infected and then dosed for an additional 21 days. Viral clearance is quantified by measuring infectious virus (plaque-forming units) at various times following infection. Dexamethasone may be used as a positive immunomodulatory control. This host resistance assay has been used in Balb/c, C57BL/6, and B6C3F1 mice and Fischer 344 (CDF), Brown Norway, and... 

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]. 

Viral selection is based on (1) relevant viruses that are actual viruses (or of the same species as actual viruses and relevant to the host cell) that have been identified as contaminants (or potential contaminants) of the process, (2) specific model viruses that are closely related to actual viruses (e.g., same genus or family) and have similar physico-chemical properties, and (3) nonspecific model viruses believed to be representative of the spectrum of different virus physio-chemical characteristics [5, 50]. Nonspecific model viruses are used to show inactivation/ removal of viruses in general and to characterize purification robustness [50]. Virus clearance studies should cover emerging viruses and viruses currently believed to be absent in raw materials. These concerns are not addressed when relying on direct testing to ensure safety, specifically consideration of future virus removal requirements in anticipation of future regulatory changes [5, 41]. 

Chromatography steps for purification of monoclonal antibody produced by CHO cells Scale-down studies for resin use and viral clearance fully predictive of larger scale [26] (Genentech)... 

Purification of a therapeutic monoclonal antibody Scale-down characterization studies for various filtration and chromatography steps (including viral clearance) to predetermine acceptance criteria [87] (Biogen Idee)... 

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