Virus inactivation methods

Nuanualsuwan, S. and Cliver, D. Q. (2002). Pretreatment to avoid positive RT-PCR results with inactivated viruses. /. Virol. Methods 104, 217-225. 

The potential of inactivated viral particles as effective vaccines has gained some attention, but again fears of accidental transmission of disease if inactivation methods are not consistently 100 per cent effective have dampened enthusiasm for such an approach. In addition, the stringent containment conditions required to produce large quantities of the virus render such production processes expensive. 

The use of animals for the preclinical evaluation of blood derivatives primarily encompassed evaluation for activity [4], and later for viral contamination, but little from the perspective of actual toxicological endpoints. Many of the hemophiliacs who were treated with early versions of antihemophilic factor (AHF) and Factor IX became infected with hepatitis [5], This provided the major reason for developing viral inactivation methods for AHF concentrates. The hepatitis agents were referred to as non-A, non-B hepatitis (NANB). The preclinical demonstration that the active virus had been inactivated required the use of chimpanzees, which were injected with the AHF concentrate. During the early 1980s plasma fractionators used chimpanzee studies to demonstrate the effectiveness for the reduction of HBV and NANB hepatitis infectivity [6], Ultimately, previously untreated patients were evaluated in clinical trials that demonstrated the utility of a number of viral reduction methods for what was by then known as hepatitis C [7],... 

During inactivation steps, viral infectivity is reduced by treatment with chemicals and/or physical methods. Remnants of virus particles (e.g., viral nucleic acids) may remain in the product-containing fraction but are not infectious. Chemical methods of virus inactivation, such as treatment with solvent-detergent or acetone, must be placed upstream, since subsequent steps are needed to remove or reduce the levels of the toxic chemicals. Terminal inactivation is often achieved using physical methods, such as heat and low pH, because these methods leave no chemical residues. After treatment, the final products are delivered to patients, so aseptic processing conditions must be maintained throughout terminal inactivation steps and the parameters for virus inactivation must be balanced with the conditions to preserve product quality and yield. 

Fig. 9 Kinetics of non-enveloped virus inactivation during 80°C heat treatment of freeze dried Factor VIII (FVIII) concentrate (A) in the presence of high cake moisture (>0.8% moisture) or (B) in the presence of low cake moisture (<0.8% moisture). Methods were as described in Fig. 8 (gray boxes = logio virus titer, closed circles = % moisture, and dashed line = virus detection limit).

Screening and selection of the source plasma will only avoid contamination by known pathogens. The protein purification steps and specific virus reduction methods used in production processes, however, will inactivate and/or remove both known and unknown viruses. Terminal virus inactivation treatments are applied to product in final container and must balance virus inactivation with any modifications to protein immunogenicity, activity, and yield. While many upstream virus inactivation steps rely on chemical methods that involve the addition and subsequent removal of toxic agents (e.g., solvent/detergent), physical methods for virus inactivation, such as pH and heat, are used for terminal steps. 

Manufacturing processes have evolved dramatically over the last few years. In the late 1980s, 76 /o of hemophiliacs were HCV positive, ° and between 1979 and 1985, approximately 50 /o of hemophiliacs had acquired HIV from plasma-derived FVIII. Since then, however, most U.S.-licensed plasma derivatives have not transmitted HBV, HCV, or HIV as a result of improvements in donor screening and test methods, and the inclusion of effective upstream virus-reduction and terminal virus-inactivation steps in manufacturing processes. Residual risks of virus transmission from plasma-derived products are now largely associated with non-enveloped viruses. " Thus, the need for additional terminal or upstream virus inactivation/removal steps still exists, but the current challenge is to develop cost effective methods against physico-chemically resistant non-enveloped viruses, such as human parvovirus B19. 

It is, however, important to consider two circumstances separately on the one hand the risk of transmitting disease with whole blood or cellular blood components under conditions where virus inactivation or virus removal methods are scarcely available, and on the other hand the far lesser risk of transmitting disease with plasma or plasma-derived products prepared in those centers where effective virus inactivation procedures can be applied to the product. 

Since viral inactivation methods, such as solvent detergent and heat pasteurization, were implemented in the production process, the risks of HIV and hepatitis have virtually been ehminated. Dry heating at 60 C is insufficient to eliminate all hepatitis C virus, which requires dry heating at 80 C, pasteurization, or treatment with mixtures of solvents and detergents (14). Nevertheless, many viral inactivation methods currently used do not completely eliminate certain (non-enveloped) viruses, for example parvovirus and hepatitis A (11,15) removal of the small, non-hpid-enveloped parvovirus B19 requires 15 nm nanofiltration. HIV appears to progress more rapidly in patients co-infected with hepatitis B and cytomegalovirus (11). In addition, hepatitis C rephcated more rapidly in patients infected with HIV (11). 

The phenomenon of viral adsorption to various surfaces was extensively studied from an environmental standpoint as reviewed by Daniels (14) and Gerba (15) for prevention of various waterborne viral transmissions. The problem of virus removal from complex protein solutions is very different from that of sewage and drinking water treatment processes because most protein molecules compete for the active sites of the adsorbents. Hence, both the adsorption rate and capacity diminish in the presence of protein molecules (16). It is the intention of this paper to demonstrate and to compare the antiviral activity of a surface-bonded QAC in aqueous solutions against 2 model viruses with and without the presence of proteins. The efficacy of the accepted antiviral thermo-inactivation was compared with the viral inactivation method by the surface-bonded QAC treatment. Beta-lactamase was used as a thermolabile model protein (17), and bacteriophage T2 and herpes simplex virus type 1 (HSV-1, an enveloped animal virus) were used as model hydrophilic and hydrophobic viruses to test these chemical inactivation methods. 

Among the different methods for virus inactivation, high-temperature short-time (HTST) has been used for retrovirus clearance, whereas ultra-violet irradiation is most powerful for the elimination of small, non-enveloped and otherwise very resistant vimses such as porcine parvo-vims (PPV) [169-171]. 

Sofer, G., Sofer, D.C., Boose, ).A., Inactivation methods grouped by virus. Virus inactivation in the 1990s - and into the 21st century. BioPharm Int. 2003, Suppl. S37-S42. 

Virus removal and inactivation have been reviewed by several authors [23, 25-27]. It is generally accepted that conventional water treatment practices can reduce viral levels by a factor of 10 to 10 in the finished water. Disinfection, mostly in the form of chlorination, has been the main method of virus inactivation in drinking water. Viruses seem to be considerably more resistant than coliforms, thus requiring higher doses and longer contact times (for ref. see [16]). 

Validation of methods used for virus removal or virus inactivation should not be conducted in the production facilities in order not to put the routine manufacture at any risk of contamination with the viruses used for validation. 

Among chemical methods, monochloramine and free chlorine are used extensively to inactivate viruses." Inactivation can also occur hy degradation or disruption of any one of the viruses structures or hy all of them, depending on the chemistiy of the disinfectant." ... 

Strancar, A., P. Raspor, H. Schwinn, R. Schiitz, D. Josic, Extraction of Triton X-100 and its determination in virus-inactivated human plasma by the solvent-detergent method, J. Chw-matogr., 1994, 658,475-481. 

Inactivation and Removal of Viruses. In developing methods of plasma fractionation, the possibiHty of transmitting infection from human vimses present in the starting plasma pool has been recognized (4,5). Consequentiy, studies of product stabiHty encompass investigation of heat treatment of products in both solution (100) and dried (101) states to estabHsh vimcidal procedures that could be appHed to the final product. Salts of fatty acid anions, such as sodium caprylate [1984-06-17, and the acetyl derivative of the amino acid tryptophan, sodium acetyl-tryptophanate [87-32-17, are capable of stabilizing albumin solutions to 60°C for 10 hours (100) this procedure prevents the transmission of viral hepatitis (102,103). The degree of protein stabilization obtained (104) and the safety of the product in clinical practice have been confirmed (105,106). The procedure has also been shown to inactivate the human immunodeficiency vims (HIV) (107). 

Heat is the most reliable method of virus disinfection. Most human pathogenic viruses are inactivated following exposure at 60°C for 30 minutes. The virus of serum hepatitis can, however, survive this temperature for up to 4 hours. Viruses are stable at low temperatures and are routinely stored at -40 to -70°C. Some viruses are rapidly inactivated by drying, others survive well in a desiccated state. Ultraviolet light inactivates viruses by damaging their nucleic acid and has been used to prepare viral vaccines. These facts must be taken into account in the storage and preparation of viral vaccines (Chapter 15). 

Duck hepatitis B virus (DHBV) has been proposed as a possible model for the inactivation of human HBV by chemical disinfectants. The principle of the test method uses viral DNA polymerase (DNAP) as a target, total inhibition in vitro of DNAP by chemical disinfectants being predictive of inactivation of infectivity in vivo. 

Baert, L., Debevere, J., and Uyttendaele, M. (2009a). The efficacy of preservation methods to inactivate foodborne viruses. Int. ]. Food Microbiol. 131, 83-94. 

Currently, all donors and blood preparations undergo multistage and expensive control to ensure the absence of viral contamination In this respect, the development of affordable methods of inactivation of viruses could be an important step toward safety in hemotransfusion. Currently used treatments such as UV irradiation damage therapeutic components of the blood (Williamson and Cardigan, 2003), so alternative selective approaches are needed for this purpose. Among them, chemotherapy, photochemotherapy (PCT), and photodynamic antibacterial therapy should be noted (Mohr, 2000). 

Mohr H (2000) Inactivation of viruses in human plasma. Methods Enzymol. 319 207-216. Wainwright M (2002) The emerging chemistry of blood product disinfection. Chem Soc Rev. 31 128-136. 

Better methods for preparing clotting factors from blood and the development of recombinant clotting factors provided the solutions. Methods of detecting, inactivating, and removing viruses were improved, and none of the hemophilia replacement products— conventional or recombinant—has transmitted either HIV or hepatitis since 1987. As an alternative, recombinant clotting factors 8 and 9, produced in animal cells, were approved in 1992 and 1997, without the risk associated with human blood products. 

Table 10.12. Methods usually employed to inactivate bacteria or viruses subsequently used as dead/inactivated vaccine preparations...

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