Viral Inactivation & Removal
Depending on the type of fraction being purified a variety of validated viral inactivation and removal steps are performed during the manufacturing of plasma derived products. Viral removal procedures such as ultrafiltration and chromatography result in a product without any virus or viral proteins in the final product. Viral inactivation procedures on the other hand, such as S/D treatment, pasteurization or caprylate acid treatment are procedures where the viruses are present in the final product but in an inactive state.
Enveloped Viral Inactivation methods
The virus inactivation methods largely target enveloped viruses by targeting the disruption of lipid envelopes surrounding the viral capsid (a protein shell that holds viral DNA or RNA). Enveloped viruses are sensitive to heat, detergents and dessication and are therefore easier to sterilize than the non-enveloped viruses.
The gold standard for enveloped virus inactivation is S/D treatment or short for solvent/ detergent treatment. The solvent solubilizes the lipid envelopes leaving the virus exposed to detergents resulting in virus inactivation. The treatment does not have any effect on the structure or bioactivity of the plasma proteins as it targets lipids and lipid derivatives only. S/D treated plasma results in a significant drop in viral load (>6 log steps) in less than 2mins [1,2]. Some of the enveloped viruses targeted include HIV-1,-2, HBV and HCV, infact some of the recently discovered viruses such as the West Nile Virus, Chickungunya Virus and SARS coronavirus are inactivated using S/D treatment .
Pasteurization utilizes elevated temperatures to denature viral proteins; therefore this viral inactivation technique is not suitable for any heat labile plasma proteins. Denaturation results in three dimensional changes in the viral protein structure making them non-functional. Pasteurization is effective against both enveloped and non-enveloped viruses since the presence or absence of a lipid coat doesn’t protect the virus from hot temperatures. Pasteurization is typically performed at 60ºC for 10 hours.
Non-Enveloped Viral Removal methods
Contamination of source plasma by enveloped viruses (HIV, HCV) in the 80’s led to the development of effective technologies against these types of viruses. However removal of the smaller non-enveloped viruses (Hepatitis A, Parvo B19 virus ) is not very effective using these techniques.
A reliable and robust industry standard today for non-enveloped virus clearance is nano-filtration. Nanofiltration is a type of ultrafiltration process where a filter of defined pore size is used to retain the viruses while the liquid plasma flows through. The pore sizes on these filters typically range between 15-40nm
With nanofiltration there is no exposure of the plasma proteins to extreme pH levels and temperature. Also the filter surface is inert and does not lead to changes in structural or chemical properties of the plasma proteins. Nanofiltration is show to reduce the ParvoB19 (35nm filter) load by >7.5 log . Recently use of nanofilters for removal of prions is being evaluated, creating new possibilities for even safer plasma derived products.
Chromatography is a method of separation where components are selectively separated based on their affinity to the stationary phase and the mobile phase. Components with affinity to the stationary phase remain attached to it, while the components with affinity to the mobile phase flow through. Separation based on differences in affinity between components in a liquid is known as affinity chromatography. There are many different types of chromatography procedures namely ion-exchange, size exclusion and partition chromatography.
The viral load clearance capacity of this technique depends upon the physiochemical and biochemical properties of the virus. Therefore validation studies using spiked samples must be performed before using chromatographic viral removal procedures.
- Biesert, L. and Shartono H. Solvent/detergent treatment of human plasma – a very robust method for virus inactivation. Validation of virus safety of Octaplas. Vox Sang. 1998;74(suppl 1):207-212
- Horowitz, B., Lazo, A., Grossberg, H., Page, G., Lippin, A., Swan, G. Virus inactivation by solvent/detergent treatment and the manufacture of SD-plasma. Vox Sang. 1998;74(suppl l):203-206.
- Hellstern, P. and Solheim, B. The use of solvent/ detergent treatment in Pathogen Reduction of Plasma. Transfus Med Hemother. 2011; 38: 65-70
- Yokoyama, T., Murai, K., Murozuka, T., Wakisaka, A., Tanifuji, M., Fujii, N., Tomono, T. Removal of small non-enveloped viruses by nanofiltration. Vox Sang. 2004; 86(4):225-9