Viral clearance

A broad and vigorons T cell response generally accompanies elimination of HBV as well as HCV infection. By contrast, patients with chronic hepatitis B or C tend to have late, transient, or narrow T cell responses. In a long-term follow-up of HBV-infected patients receiving HPC transplants from HBV-immune individuals, 20 of 31 recipients cleared their HBV infection (Hui et al. 2005). In principle, these results encourage the development of adoptive T cell transfer strategies for the treatment of chronic viral hepatitis. However, it is still controversial whether induction of an efficient T cell response is the cause or the consequence of viral clearance. Furthermore, T cell responses do not only contribute to virus control but also to disease pathology (Rehermann and Nascimbeni 2005). 

Decreased macrophage infiltration and demyelination Decreased CD4+ T cell trafficking into the CNS, impaired viral clearance No effect on CD8+ T cell trafficking into the CNS Enhanced fatality with delayed CD 8+ T cell infiltration No effect CCR5A32 not protective... 

Neff-LaFord, H., Vorderstrasse, B., and Lawrence, B.R, Fewer CTL, not enhanced NK cells, are sufficient for viral clearance from the lungs of immunocompromised mice, Cell. Immunol., 226, 54, 2003. 

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. 

These include, but are not limited to, cGMP and regulatory compliance issues such as the need to provide proper segregation of pre- and post-viral clearance steps, ensure uni-directional flow of material and personnel, cleaning of the facility, waste handling, and environmental monitoring of the facility (37,57). In order to scale-up and transfer a process successfully from laboratory scale to pilot scale and multiple commercial manufacturing scales. 

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]. 

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. 

Regardless of which assays are used, there are many variables that must be controlled during viral clearance evaluation. Some of these are listed in Table 6. 

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

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. 

Darling, A. J. Validation of the purification process for viral clearance evaluation. In G. Sofer, D. Zabriskie, eds. Biopharmaceutical Process Validation. New York Marcel Dekker (2000). 

Aranha, H. (2001, January). Viral clearance strategies for biopharma-ceutical safety, Part 1 general Considerations, BioPharm, pp. 28—35. 

It is produced in recombinant CHO cells cultured in a medium free of animal-derived components. The BeneFix production process involves an ultrafiltration/diafiltration step, followed by four chromatographic steps ion exchange (Q resin), pseudo-affinity (Cellufine sulfate resin), hydroxyapatite, and affinity (immobilized Cu2+ ions). After these chromatographic steps, there are membrane processes (nanofiltration for viral clearance and diafiltration for solvent exchange), after which the purified protein is formulated (Edwards and Kirby, 1999). 

The risk of viral contamination is a feature common to all biotechnology products derived from cell lines. In this session, we heard several talks related to viral contamination and viral clearance. 

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