Viral processing
Encyclopedia
The main idea behind viral processing is to stop the viruses in a given sample from infecting the desired product. The two most widely used methods of viral processing are viral removal and viral inactivation. The former is a method in which all viruses are simply removed from the sample completely. The latter method is one in which the viruses may remain in the final product, but in a non-infective form. These techniques are used widely in the food and blood plasma
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 parvovirus
es. The methods used in the plasma industry have been summarized (Horowitz B., Minor P., Morgenthaler J. J., Burnouf T., McIntosh R., Padilla A., Thorpe R. and van Aken W. G. Who Expert Committee on Biological Standardization. World Health Organ Tech Rep Ser. 924: 1-232, 2004.) In some cases, however, it is the virus itself that is the desired product, as is often the case with the HIV. In many cases, researchers may be trying to extract the viruses from the blood for study, not specifically for blood purification. It is also common to use these types of techniques to remove particles produced as a result of viral infection.
or filtration
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.
. 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 15 , VAG - 300 , Viresolve 180 , and Viresolve 70TM.
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 affects on the process stream or the product itself.
, 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
http://www.redcross.org/. 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 live without their lipid coating, so they die when exposed to these detergents. Other viruses may still live, 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.
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.
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
.
, 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.
UV light in combination with riboflavin has been shown to be effective in reducing pathogens in blood transfusion products.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.
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.
Ri = -log(Yi) ± 1
where Ri is the reduction factor, Yi is the ratio of beginning viral count/activity to ending viral count/activity and the plus or minus 1 is a correction factor for the one order of magnitude error in the calculation.
of the ratio of the beginning viral number to the ending viral number plus or minus one, and is used as an indicator as to whether or not a specific process is appropriate for the processing of a certain virus under certain conditions. The reduction factor needed for a certain process stream is dependent on many different factors, some of which include:
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
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
Hepatitis
Hepatitis is a medical condition defined by the inflammation of the liver and characterized by the presence of inflammatory cells in the tissue of the organ. The name is from the Greek hepar , the root being hepat- , meaning liver, and suffix -itis, meaning "inflammation"...
A, B, and C; and parvovirus
Parvovirus
Parvovirus, often truncated to "parvo", is both the common name in English casually applied to all the viruses in the Parvoviridae taxonomic family, and also the taxonomic name of the Parvovirus genus within the Parvoviridae family...
es. The methods used in the plasma industry have been summarized (Horowitz B., Minor P., Morgenthaler J. J., Burnouf T., McIntosh R., Padilla A., Thorpe R. and van Aken W. G. Who Expert Committee on Biological Standardization. World Health Organ Tech Rep Ser. 924: 1-232, 2004.) In some cases, however, it is the virus itself that is the desired product, as is often the case with the HIV. In many cases, researchers may be trying to extract the viruses from the blood for study, not specifically for blood purification. It is also common to use these types of techniques to remove particles produced as a result of viral infection.
Virus 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 extractionExtraction
Extraction may refer to:* Extraction , an album by guitarist Greg Howe* Extraction , the separation of a substance from a matrix* Extraction , the removal of someone from a hostile area to a secure location...
or filtration
Filtration
Filtration is commonly the mechanical or physical operation which is used for the separation of solids from fluids by interposing a medium through which only the fluid can pass...
methods. Some of the more prominent methods include:
- Nanofiltration
- ChromatographyChromatographyChromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
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 remove viruses specifically by size exclusion. This type of process is typically used for parvoviruses and other viruses containing a protein coatCapsid
A capsid is the protein shell of a virus. It consists of several oligomeric structural subunits made of protein called protomers. The observable 3-dimensional morphological subunits, which may or may not correspond to individual proteins, are called capsomeres. The capsid encloses the genetic...
. 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
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
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 15 , VAG - 300 , Viresolve 180 , and Viresolve 70TM.
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. It is also worthy to note that the effectiveness of this process can vary greatly between viruses and that the efficiency of the process can change based on the buffer that is used. Sanitation between batches is also a concern when performing this procedure.Viral inactivation
Viral inactivation renders viruses inactive, or unable to infect. Many viruses contain lipidLipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...
or protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
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
Denaturation (biochemistry)
Denaturation is a process in which proteins or nucleic acids lose their tertiary structure and secondary structure by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent , or heat...
the virus completely. Viral inactivation is used widely in the blood plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
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:
- Solvent/detergent inactivation
- PasteurizationPasteurizationPasteurization is a process of heating a food, usually liquid, to a specific temperature for a definite length of time, and then cooling it immediately. This process slows microbial growth in food...
(heating) - Acidic pH inactivation
In some cases viral inactivation is not a viable removal alternative because even the denatured or otherwise inactivated viral particles can have deleterious affects on the process stream or the product itself.
Solvent/detergent (S/D) inactivation
This process, developed by the New York Blood CenterNew York Blood Center
New York Blood Center bills itself as the "nation's largest, community-based, non-profit, independent blood center." Founded in 1964, it relies upon a staff of 2,000 workers and a much smaller permanent staff in order to supply over 200 hospitals in New York and New Jersey with a source of whole...
, 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
American Red Cross
The American Red Cross , also known as the American National Red Cross, is a volunteer-led, humanitarian organization that provides emergency assistance, disaster relief and education inside the United States. It is the designated U.S...
http://www.redcross.org/. This process is only effective for viruses enveloped in a lipid
Lipid
Lipids constitute a broad group of naturally occurring molecules that include fats, waxes, sterols, fat-soluble vitamins , monoglycerides, diglycerides, triglycerides, phospholipids, and others...
coat, however. The detergents used in this method interrupt the interactions between the molecules in the virus's lipid coating. Most enveloped viruses cannot live without their lipid coating, so they die when exposed to these detergents. Other viruses may still live, 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.
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 pasteurizationPasteurization
Pasteurization is a process of heating a food, usually liquid, to a specific temperature for a definite length of time, and then cooling it immediately. This process slows microbial growth in food...
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
Celsius
Celsius is a scale and unit of measurement for temperature. It is named after the Swedish astronomer Anders Celsius , who developed a similar temperature scale two years before his death...
.
Acidic pH inactivation
Some viruses, when exposed to a low pHPH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
, 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.Riboflavin and UV light
Pathogen reduction using riboflavin and UV light
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,...
damages the nucleic acids in viruses, bacteria, parasites, and donor white blood cells rendering them unable to replicate and cause disease.
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. 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 the ratio of these two values is calculated. This value is used to determine the reduction factor of the process using the following equation:Ri = -log(Yi) ± 1
where Ri is the reduction factor, Yi is the ratio of beginning viral count/activity to ending viral count/activity and the plus or minus 1 is a correction factor for the one order of magnitude error in the calculation.
Reduction factor
The reduction factor is defined as the negative logLogarithm
The logarithm of a number is the exponent by which another fixed value, the base, has to be raised to produce that number. For example, the logarithm of 1000 to base 10 is 3, because 1000 is 10 to the power 3: More generally, if x = by, then y is the logarithm of x to base b, and is written...
of the ratio of the beginning viral number to the ending viral number plus or minus one, and is used as an indicator as to whether or not a specific process is appropriate for the processing of a certain virus under certain conditions. The reduction factor needed for a certain process stream is dependent on many different factors, some of which include:
- The expected initial concentration of virus
- The product being purified
- The infective dose of the virus (for in vivoIn vivoIn vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
usage) - Whether inactivation is a viable alternative to complete removal
- The capabilities of the laboratory
- The relative difficulty of the inactivation or removal method
Applications
This technology has been used extensively in the food and drug industries, but some other applications of viral processing have been:- Air purification (Millipore)
- VaccineVaccineA vaccine is a biological preparation that improves immunity to a particular disease. A vaccine typically contains an agent that resembles a disease-causing microorganism, and is often made from weakened or killed forms of the microbe or its toxins...
s - Viral sample extraction
- Water purification
- West Nile Virus inactivation