Viruses clearance

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

Byrne, J.A. and Oldstone, M.B., Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus clearance of virus in vivo, /. Virol., 51, 682-686,1984. 

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. 

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

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],... 

The principle is to have about three steps the first with specific properties to capture the antibody from the crude feedstock, the second to separate impurities, and the third to play a polishing role with properties to remove traces of peculiar agents such as nucleic acids and endotoxins, and able to add virus clearance. 

An example of virus clearance factors in chromatographic processes frequently used for purification of antibodies is given in Table 17. Lower clearance factors for protein A affinity chromatography have been found by Mariani and Tarditi128 when compared to results found with protein G by Walter and Allgaier.237 An explanation of this fact can be found in the fact that protein G requires harsher elution conditions than protein A. 

Bekkering, RC., Brouwer, J.T., Leroux-Roels, G., van Vlierberghe, H., Elewaut, A., Schalm, S.W Ultrarapid hepatitis C virus clearance by daily high-dose interferon in non-responders to standard therapy. J. Hepatol. 1998 28 960-964... 

Pasture, G., Monno, L., Santantonlo, T., Angavano, G., Milella, M., GiannelU, A., Fiore, J.R. Hepatitis B virus clearance from serum and Uver after acute hepatitis delta superinfection in chronic HBsAg carriers. X Med. Virol. 1990 31 284-290... 

Parkkinen, J. et al., A modified capryhc acid method for manufacturing immunoglobuhn G from human plasma with high yield and efficient virus clearance. Vox Sang., 90, 97-104, 2006. 

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 discussed here are for Plasbumin -25, which is purified from Fraction V paste and consists of 23.5 /o-26.5 /o protein, of which no less than 96.5%o is albumin. The preparation is stabilized with 0.02 M sodium caprylate and 0.02 M acetyltryptophan and contains 145mEq/L sodium. For the virus clearance experiments, virus was spiked into albumin, pH 6.4-7.4, and the solution was heated at 60°C for 10 hr. Aliquots for virus titration were removed as soon as virus was added (preheat), when the temperature of the solution reached 60° C (Ohr) and at various times during the pasteurization cycle. Unheated albumin and HBSS were also spiked to the same dilution with virus and tested as positive controls. 

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... 

A process step with many variables to control is equally difficult to scale down for bench scale studies. However, results from animal studies and human clinical trials have been consistent with the in vitro model virus clearance studies of the terminal freeze dry/dry heat treatment. 

Virus clearance studies of Factor VIII (Hemofil T) indicated the freeze dry/dry heat treatment (60°C, 72 hr) step was effective in inactivating... 

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. 

Perhaps more difficult to duplicate or control during small scale virus clearance studies, are the precise... 

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. 

Certain process steps may be easier to model than others and duplicating the freeze dry/dry heat step at small scale is very difficult. The conclusions drawn from virus clearance studies are reliable only when the appropriateness of the small-scale models can be demonstrated. During the freeze dry/dry heat step of Koate -DVI, virus reduction was dependent on moisture levels so even the formulation of stoppers, which could impact the absorption of water during autoclaving and its release to freeze dried material, must be considered and tested. 

Murray JM, Wieland SE, Purcell RH, Chisari EV (2005) Dynamics of hepatitis B virus clearance in chimpanzees. Proc Natl Acad Sd USA 102 17780-17785 Ni Y-H, Chang M-H, Huang L-M, Chen HL, Hsu HY, Chiu TY et al (2001) Hepatitis B virus infection in children and adolescents in a hyperendemic area 15 years after mass hepatitis B vaccination. Ann Intern Med 135 796-800... 

Ilback, N.G., L. Wesslen, J. Fohbnan, and G. Friman. 1996. Effects of methyl mercury on cytokines, inflammation and virus clearance in a common infection (coxsackie B3 myocarditis). Toxicol. Lett. 89(1) 19-28. 

Validation of the virus clearance capability of the production process. 

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]. 

Virus loads in unprocessed, prepurified bulk (harvest samples) are quantified, typically via transmission by electron microscopy or infectivity, to estimate actual virus load versus the expected virus removal capacity of the process [47,50]. Total virus clearance should exceed measured levels by at least 3-5 logs [47]. An example of a typical virus load is 10 to 10 RVLPs (retrovirus-like particles) per milliliter of unprocessed bulk from CHO cells [3]. Clearance factors of 15-20 logs are typical for murine retroviruses [3] and slightly more than for lipid-enveloped viruses. For most viruses besides endogenous retroviruses, however, there is no reasonable way to establish virus load in the bulk because these types of viruses should not normally be present. In addition direct testing methods are limited inherently because they are designed to detect only known and not known contaminants [5]. Thus, the... 

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]. 

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... 

Taylor RP, Sutherland WM, Martin EN, Ferguson PJ, Reinagel, ML, Gilbert E, Lopez K, Incardona NL, Ochs HD. 1997. Bispecific monoclonal antibody complexes bound to primate erythrocyte complement receptor 1 facilitiate virus clearance in a monkey model. The Journal of Immunology 158 842-850. 

It appears that the mechanisms of CXCL9- and CXCLlO-mediated viral clearance involve NK cells and IFNs. The increases in lymphoid cell numbers at foci of infection contribute to virus clearance through the action of cytolysis and IFN secretion. Through the induction... 

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