Virus inactivation

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Viral inactivation is to stop the viruses in a given sample from contaminating the desired product either by removing viruses completely or rendering them non-infectious. These techniques are used widely in the food and blood plasma [1] industries, as those products can be harmed by the presence of viral particles. Some of the more common viruses removed by these methods are the HIV-1 and HIV-2 viruses; hepatitis A, B, and C; and parvoviruses. [2] These methods have been adapted to remove prions, which are not related to viruses, from blood products. [3]

Contents

Removal

This overarching process, which has come to be known simply as virus removal, is one in which all of the viruses in a given sample are removed by traditional extraction or [full energy] methods. Some of the more prominent methods include:

These extraction processes are considered "traditional processes" because they do not chemically affect the virus in any way; they simply remove it physically from the sample.

Nanofiltration

Virus removal processes using nanofiltration techniques [4] remove viruses specifically by size exclusion. This type of process is typically used for parvoviruses [5] and other viruses containing a protein coat. A typical HIV virion is 180 nm and a typical parvovirus can vary between 15 and 24 nm, which is very small. One great advantage of filtration, as opposed to methods involving extremes of temperature or acidity, is that filtration will not denature the proteins in the sample. Nanofiltration is also effective for most types of proteins. Since it is not chemically selective, no matter what the surface chemistry of the viral particle is, viral removal processes using nanofiltration techniques will still be effective. Another great advantage of this technique is its ability to be performed on a lab scale and then effectively scaled up to production standards. It is important to consider, however, the fact that the level of removal of the viruses is dependent on the size of the pores of the nanofilter. In some cases, very small viruses will not be filtered out. It is also necessary to consider the possible effects of pressure and flow rate variation.

Some of the filters used for to perform these types of processes are Planova 15N, [6] Planova 20N, BioEX, VAG - 300, Viresolve 180, [7] Viresolve 70TM, and the Virosart [8] range.

Chromatography

Chromatographic methods of removing viruses are great for purifying the protein and are also effective against all types of viruses, but the level of virus removal is dependent on the column composition and the reagents that are used in the process. The effectiveness of this process can vary greatly between viruses and its efficiency can change based on the buffer used. Sanitation between batches is also a concern when performing this procedure.

Membrane chromatography is increasingly popular for virus purification and removal.

Inactivation

Viral inactivation renders viruses unable to infect. Many viruses contain lipid or protein coats that can be inactivated by chemical alteration. Viral inactivation is different from viral removal because, in the former process, the surface chemistry of the virus is altered and in many cases the (now non-infective) viral particles remain in the final product. Rather than simply rendering the virus inactive, some viral inactivation processes actually denature the virus completely. Viral inactivation is used widely in the blood plasma industry.

In order to achieve inactivation of the viruses in the sample, it is necessary to perform "special" purification processes that will chemically alter the virus in some way. Some of the more widely used processes are as follows:

In some cases viral inactivation is not a viable removal alternative because even the denatured or otherwise inactivated viral particles can have deleterious effects on the process stream or the product itself.

Solvent/detergent (S/D) inactivation

This process, developed by the New York Blood Center, [9] is the most widely used viral inactivation method to date. It is predominantly used in the blood plasma industry, by over 50 organizations worldwide and by the American Red Cross . This process is only effective for viruses enveloped in a lipid coat, however. The detergents used in this method interrupt the interactions between the molecules in the virus's lipid coating. Most enveloped viruses cannot exist without their lipid coating so are destroyed when exposed to these detergents. Other viruses may not be destroyed but they are unable to reproduce rendering them non-infective. The solvent creates an environment in which the aggregation reaction between the lipid coat and the detergent happen more rapidly. The detergent typically used is Triton X-100.

Chemical Structure of Triton X-100 (n = 9-10). Triton X-100.svg
Chemical Structure of Triton X-100 (n = 9-10).

This process has many of the advantages of the "traditional" removal techniques. This process does not denature proteins, because the detergents only affect lipids and lipid derivatives. There is a 100% viral death achieved by this process and the equipment is relatively simple and easy to use. Equipment designed to purify post-virus inactivated material would be necessary to guard against contamination of subsequent process streams.

S/D treatment utilizes readily available and relatively inexpensive reagents, but these reagents must be removed from the product prior to distribution which would require extra process steps. Because this process removes/inactivates the lipid coating of a virus, viruses without any sort of lipid envelope will be unaffected. There is also no inactivation effect by the buffers used in this process.

Pasteurization

Inactivation of viruses by means of pasteurization can be very effective if the proteins that you are trying to protect are more thermally resistant than the viral impurities with which they are in solution. Some of the more prominent advantages of these types of processes are that they require simple equipment and they are effective for both enveloped and non-enveloped viruses. Because pasteurization involves increasing the temperature of solution to a value that will sufficiently denature the virus, it does not matter whether the virus has an envelope or not because the envelope alone cannot protect the virus from such high temperatures. However, there are some proteins which have been found to act as thermal stabilizers for viruses. Of course, if the target protein is not heat-resistant, using this technique could denature that target protein as well as the viral impurity. Typical incubation lasts for 10 hours and is performed at 60°C.

Acidic pH inactivation

Some viruses, when exposed to a low pH, will denature spontaneously. Similar to pasteurization, this technique for viral inactivation is useful if the target protein is more resistant to low pHs than the viral impurity. This technique is effective against enveloped viruses, and the equipment typically used is simple and easy to operate. This type of inactivation method is not as effective for non-enveloped viruses however, and also requires elevated temperatures. So in order to use this method, the target protein must be resistant to low pHs and high temperatures which is unfortunately not the case for many biological proteins. Incubation for this process typically occurs at a pH of 4 and lasts anywhere between 6 hours and 21 days.

Ultraviolet (UV) inactivation

UV rays can damage the DNA of living organisms by creating nucleic acid dimers. However, the damages are usually not important due to low penetration of UVs through living tissues. UV rays can be used, however, to inactivate viruses since virus particules are small and the UV rays can reach the genetic material, inducing the dimerisation of nucleic acids. Once the DNA dimerised, the virus particules cannot replicate their genetic material which prevent them from spreading.

UV light in combination with riboflavin has been shown to be effective in reducing pathogens in blood transfusion products. [10] [11] Riboflavin and UV light damages the nucleic acids in viruses, bacteria, parasites, and donor white blood cells rendering them unable to replicate and cause disease. [12] [13] [14]

Spiking studies

In many cases, the concentration of viruses in a given sample is extremely low. In other extraction processes, low levels of impurity may be negligible, but because viruses are infective impurities, even one viral particle may be enough to ruin an entire process chain. It is for this reason that special measures must be taken to determine the appropriate removal or inactivation method for whatever type of virus is being extracted from whatever type of solution.

Spiking studies were created specifically for this purpose. A spiking study is a study done in order to determine the possible methods of viral removal or inactivation. The results of these studies are numerical and, based on these numbers, researchers can determine whether or not the process on which the study was conducted will be suitable for the viruses they are trying to extract and the solution from which they are trying to extract them.

The method

It has been shown through experimentation, that increasing the viral count (or level of activity) of a sample by a factor of 104 or 105 of the original will only change the virus removal/inactivation ratios by one order of magnitude [reference?]. From this knowledge, spiking studies have been created in which the virus number (or level of activation) is increased or "spiked" by a factor of 104 or 105 of the original sample. This new high number or level of activity is then run through the process stream and purified. The number or level of activity is taken at the beginning and at the end of the process stream and used in the calculation of Reduction Factor.

Reduction factor

Reduction factor (RF) for a virus removal or inactivation step is calculated using the following equation: [15]

RFstep = log10 [(V1 x T1)/(V2 x T2)]

Where: V1 = volume of spiked feedstock prior to the clearance step; T1 = virus concentration of spiked feedstock prior to the clearance step; V2 = volume of material after the clearance step; and T2 = virus concentration of material after the clearance step.

The reduction factor needed for a certain process stream is dependent on many different factors, some of which include:

Applications

This technology has been used extensively in the food and drug industries, but some other applications of viral processing have been:

Related Research Articles

Sodium dodecyl sulfate (SDS) or sodium lauryl sulfate (SLS), sometimes written sodium laurilsulfate, is an organic compound with the formula CH3(CH2)11OSO3Na and structure H3C−(CH2)11−O−S(=O)2−O−Na+. It is an anionic surfactant used in many cleaning and hygiene products. This compound is the sodium salt of the 12-carbon organosulfate. Its hydrocarbon tail combined with a polar "headgroup" give the compound amphiphilic properties that make it useful as a detergent. SDS is also component of mixtures produced from inexpensive coconut and palm oils. SDS is a common component of many domestic cleaning, personal hygiene and cosmetic, pharmaceutical, and food products, as well as of industrial and commercial cleaning and product formulations.

<span class="mw-page-title-main">Polyacrylamide gel electrophoresis</span> Analytical technique

Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation, and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.

<span class="mw-page-title-main">Blood-borne disease</span> Medical condition

A blood-borne disease is a disease that can be spread through contamination by blood and other body fluids. Blood can contain pathogens of various types, chief among which are microorganisms, like bacteria and parasites, and non-living infectious agents such as viruses. Three blood-borne pathogens in particular, all viruses, are cited as of primary concern to health workers by the CDC-NIOSH: HIV, hepatitis B (HVB), & hepatitis C (HVC).

Lysis is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a lysate. In molecular biology, biochemistry, and cell biology laboratories, cell cultures may be subjected to lysis in the process of purifying their components, as in protein purification, DNA extraction, RNA extraction, or in purifying organelles.

A lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze the labile macromolecules of the cells. Most lysis buffers contain buffering salts and ionic salts to regulate the pH and osmolarity of the lysate. Sometimes detergents are added to break up membrane structures. For lysis buffers targeted at protein extraction, protease inhibitors are often included, and in difficult cases may be almost required. Lysis buffers can be used on both animal and plant tissue cells.

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.

<span class="mw-page-title-main">Plasmapheresis</span> Removal, treatment and return of blood plasma

Plasmapheresis is the removal, treatment, and return or exchange of blood plasma or components thereof from and to the blood circulation. It is thus an extracorporeal therapy, a medical procedure performed outside the body.

The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. The purified DNA can then be used for downstream applications such as PCR, sequencing, or cloning. Currently, it is a routine procedure in molecular biology or forensic analyses.

Lentivirus is a genus of retroviruses that cause chronic and deadly diseases characterized by long incubation periods, in humans and other mammalian species. The genus includes the human immunodeficiency virus (HIV), which causes AIDS. Lentiviruses are distributed worldwide, and are known to be hosted in apes, cows, goats, horses, cats, and sheep as well as several other mammals.

<span class="mw-page-title-main">Triton X-100</span> Chemical compound

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<span class="mw-page-title-main">Viral envelope</span> Outermost layer of many types of the infectious agent

A viral envelope is the outermost layer of many types of viruses. It protects the genetic material in their life cycle when traveling between host cells. Not all viruses have envelopes. A viral envelope protein or E protein is a protein in the envelope, which may be acquired by the capsid from an infected host cell.

The Cohn process, developed by Edwin J. Cohn, is a series of purification steps with the purpose of extracting albumin from blood plasma. The process is based on the differential solubility of albumin and other plasma proteins based on pH, ethanol concentration, temperature, ionic strength, and protein concentration. Albumin has the highest solubility and lowest isoelectric point of all the major plasma proteins. This makes it the final product to be precipitated, or separated from its solution in a solid form. Albumin was an excellent substitute for human plasma in World War Two. When administered to wounded soldiers or other patients with blood loss, it helped expand the volume of blood and led to speedier recovery. Cohn's method was gentle enough that isolated albumin protein retained its biological activity.


Depyrogenation refers to the removal of pyrogens from solutions, most commonly from injectable pharmaceuticals.

Chromatography is a physical method of separation that distributes the components you want to separate between two phases, one stationary, the other moving in a definite direction. Cold ethanol precipitation, developed by Cohn in 1946, manipulates pH, ionic strength, ethanol concentration and temperature to precipitate different protein fractions from plasma. Chromatographic techniques utilise ion exchange, gel filtration and affinity resins to separate proteins. Since the 1980s it has emerged as an effective method of purifying blood components for therapeutic use.

Stain removal is the process of removing a mark or spot left by one substance on a specific surface like a fabric. A solvent or detergent is generally used to conduct stain removal and many of these are available over the counter.

Blood plasma fractionation are the general processes separating the various components of blood plasma, which in turn is a component of blood obtained through blood fractionation. Plasma-derived immunoglobulins are giving a new narrative to healthcare across a wide range of autoimmune inflammatory diseases.

Pascalization, bridgmanization, high pressure processing (HPP) or high hydrostatic pressure (HHP) processing is a method of preserving and sterilizing food, in which a product is processed under very high pressure, leading to the inactivation of certain microorganisms and enzymes in the food. HPP has a limited effect on covalent bonds within the food product, thus maintaining both the sensory and nutritional aspects of the product. The technique was named after Blaise Pascal, a 17th century French scientist whose work included detailing the effects of pressure on fluids. During pascalization, more than 50,000 pounds per square inch may be applied for approximately fifteen minutes, leading to the inactivation of yeast, mold, vegetative bacteria, and some viruses and parasites. Pascalization is also known as bridgmanization, named for physicist Percy Williams Bridgman.

Pathogen reduction using riboflavin and UV light is a method by which infectious pathogens in blood for transfusion are inactivated by adding riboflavin and irradiating with UV light. This method reduces the infectious levels of disease-causing agents that may be found in donated blood components, while still maintaining good quality blood components for transfusion. This type of approach to increase blood safety is also known as “pathogen inactivation” in the industry.

Protein adsorption refers to the adhesion of proteins to solid surfaces. This phenomenon is an important issue in the food processing industry, particularly in milk processing and wine and beer making. Excessive adsorption, or protein fouling, can lead to health and sanitation issues, as the adsorbed protein is very difficult to clean and can harbor bacteria, as is the case in biofilms. Product quality can be adversely affected if the adsorbed material interferes with processing steps, like pasteurization. However, in some cases protein adsorption is used to improve food quality, as is the case in fining of wines.

CLARITY is a method of making tissue transparent using acrylamide-based hydrogels built from within, and linked to, the tissue, and as defined in the initial paper, represents "transformation of intact biological tissue into a hybrid form in which specific components are replaced with exogenous elements that provide new accessibility or functionality". When accompanied with antibody or gene-based labeling, CLARITY enables highly detailed pictures of the protein and nucleic acid structure of organs, especially the brain. It was developed by Kwanghun Chung and Karl Deisseroth at the Stanford University School of Medicine.

References

  1. ↑ Shander A, Lobel GP, Javidroozi M (June 2016). "Transfusion practices and infectious risks". Expert Review of Hematology. 9 (6): 597–605. doi:10.1586/17474086.2016.1164593. PMID   26959944. S2CID   36732554.
  2. ↑ Di Minno G, Perno CF, Tiede A, Navarro D, Canaro M, Güertler L, Ironside JW (January 2016). "Current concepts in the prevention of pathogen transmission via blood/plasma-derived products for bleeding disorders". Blood Reviews. 30 (1): 35–48. doi:10.1016/j.blre.2015.07.004. PMC   7115716 . PMID   26381318.
  3. ↑ Turner ML (2018). "Safety of blood, blood derivatives, and plasma-derived products". Human Prion Diseases. Handbook of Clinical Neurology. Vol. 153. pp. 463–472. doi:10.1016/B978-0-444-63945-5.00026-X. ISBN   978-0-444-63945-5. PMID   29887153.
  4. ↑ "Nanofiltration". Eurodia.com. Retrieved 2010-11-23.
  5. ↑ "The Big Picture Book of Viruses - Parvoviruses". Virology.net. Retrieved 2010-11-23.
  6. ↑ "Planova: Home". Asahi-kasei.co.jp. 2010-07-29. Retrieved 2010-11-23.
  7. ↑ "Viresolve Process Area Module". Millipore. Retrieved 2010-11-23.
  8. ↑ "Sartorius AG Microsites: Virosart". microsite.sartorius.com. Retrieved 2016-05-24.
  9. ↑ "New York Blood Center". Nybloodcenter.org. Retrieved 2010-11-23.
  10. ↑ Ruane PH, et al., "Photochemical Inactivation of Selected Viruses and Bacteria in Platelet Concentrates Using Riboflavin and Light." Transfusion 2004; 44: 877-885.
  11. ↑ de Cock, et al.,"The Mirasol Evaluation Program: Use of Mirasol PRT for Platelets in Routine Clinical Practice>" Transfusion 2008: 48 (Suppl.):156A
  12. ↑ Goodrich RP, et al., Chapter 5: "The Antiviral and Antibacterial Properties of Riboflavin and Light: Applications to Blood Safety and Transfusion Medicine." Flavins: Photochemistry and Photobiology, Vol. 6, 2006, Royal Society of Chemistry; Cambridge, United Kingdom. E Silva and AM Edwards, editors.
  13. ↑ Kumar V, et al., "Riboflavin and UV-Light Based Pathogen Reduction: Extent and Consequence of DNA Damage at the Molecular Level." Photochemistry and Photobiology2004; 80: 15-21.
  14. ↑ Goodrich, RP, et al, "Defining 'Adequate' Pathogen Reduction Performance for Transfused Blood Components." Accepted for publication Transfusion 2010
  15. ↑ Note for Guidance on Virus Validation Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses, EMEA CPMP BWP, 268/95 1996
  16. ↑ "Optimizing Virus Filter Performance with Prefiltration". Millipore. Retrieved 2010-11-23.
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