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Biotech-Catalog Viral Clearance Studies |
Viral Clearance Studies | | Successful biosafety testing programs employ studies that evaluate the ability of the purification process to remove or inactivate any adventitious agents that may be present in the starting material. The potential for contamination with infectious agents comes from many sources, including cell substrates or other biological starting materials, cell culture supplements or production materials, or accidental contamination during production. These adventitious agents are typically viruses, but may also include bacteria, fungi, and mycoplasma. To assure safety, it is necessary for manufacturers to demonstrate that biopharmaceutical products are, in all probability, free of viral contamination. As suggested in the U.S. Food and Drug Administration "Points to Consider" documents, as well as the International Conference on Harmonisation (ICH) guidelines, this is most commonly done by first attempting to determine the risk for potential contaminating viruses by testing with state-of-the-art techniques; and second by performing validation studies in which appropriate processing steps are challenged with high titers of infectious agents (identified contaminants or appropriate models), to demonstrate the ability of the purification process to remove or inactivate virus. Each process step tested is evaluated by using sensitive and validated detection assays to measure the amount of agent removed or inactivated. Regulatory agencies worldwide mandate that viral clearance studies need to be performed at the clinical and market regulatory submission stages of product development. The following section discusses such clearance studies, with a consideration of risk assessment, design principles, agent selection, and limitations. In this section | | Study Design | Process validation involves challenging (or "spiking") the starting material of appropriate process steps with high titer infectious virus or other microbial agent and demonstrating that the process or procedure can remove or inactivate the infectious agent. Process steps are usually scaled down from production scale to bench scale and performed at an off-site location to avoid contamination in the manufacturing facility. The total virus or microbial reduction is calculated as the sum of the individual log10 reduction factors determined for each step, as long as the steps operate by independent mechanisms. Risk Assessment / Factors to Consider Before proceeding with a viral or microbial clearance study, a risk assessment should be conducted. The overall risk of virus contamination varies, depending on the product, and involves several factors that are discussed below. It is important to remember that the specific requirement for log10 clearance for each product is determined on a "case by case" basis. Starting Material
Starting materials present sample dependent (variable) risk of contamination with viruses that could be potentially infectious to humans. For example, a product derived from a prokaryotic expression system is less likely to contain a human pathogen than a product derived from human source material. Dosage Size / Frequency
If the product is administered in a single, small volume dose, the risk of the dosing regime containing a viral contaminant is lower than with products administered in multiple, large volume doses. Product Indication
The product indication – and associated risk/benefit – is an important factor that must be taken into consideration when designing a clearance study. If the product is used to treat a young patient with a non-life-threatening disease, the potential long-term effects of viral contamination require serious consideration. In contrast, a product employed to increase the quality of life during a terminal illness is often given different consideration. Although the risk of viral contamination is theoretically the same, provided that the starting material and dosing risks are identical, products used to treat non-life-threatening diseases may be considered high risk due to the potential long-term consequences.
Design Components of a Clearance Study
A viral clearance study typically is comprised of multiple, well-controlled laboratory tests to measure overt toxicity, interference with assay performance and the inactivation/removal of the virus. Microbial agent clearance studies typically only require performing the inactivation/removal steps. The following is an introduction and explanation of each section: Toxicity
This section begins with a brief introduction to the basic assay system used to detect virus, followed by the introduction and rationale for conducting toxicity studies. Virus quantitation is conducted by placing dilutions of material containing virus onto healthy cells. Virus replicates in the cells and produces a visible effect that can be measured. By testing a sufficient number of dilutions, one can accurately determine the total number of infectious virus particles present. In general, this type of viral assay is used to measure virus inactivation/removal at various steps in a manufacturing process. As a result, it is essential to have healthy and viable cells to conduct the viral clearance studies. In a toxicity study, we are looking for effects of the client sample on cells grown in culture. Dilutions of the client sample material are placed onto cells in culture (without virus). Subsequently, the cells are examined to determine if they have been destroyed (overt toxicity) or remain intact and look healthy (non-toxic). This test is of critical importance because viral clearance studies cannot be conducted with concentrations of client sample material that are toxic to cells. Broad ranges of client sample dilutions are evaluated in order to quantitate any detectable toxicity and identify concentrations where valid test results can be obtained. It is important to recognize that specific formulation or process constituents (e.g., solvent / detergent) can cause significant toxicity. As toxicity affects the sensitivity of the assay system, and potentially compromises the usefulness of the treatment step, our viral clearance experts will work with you to identify methods to reduce the effects of toxicity and increase the potential for virus reduction in your treatment step. Interference
Clearly, interference studies should be conducted before proceeding with the inactivation / removal portion of the overall viral clearance program. The generation of data that detects and quantifies interference is a critical component of viral clearance studies as it defines the test parameters where virus inactivation/clearance results will be valid. The conditions under which an interference study is conducted should mimic the samples generated in the viral removal / inactivation portion of the study. Therefore, the test article and virus should be exposed to the assay system concurrently. When interference is observed, it is commonly detected at low virus concentrations, however, there are exceptions to this statement such as the presence of neutralizing antibody to virus in a product derived from human plasma. When detected, it is critical to assess interference in test samples with low virus titers that will be used to calculate the final log10 reduction values for the individual steps. Once the toxicity study is complete, the concentrations of the sample material that are not overtly toxic must be tested further to make sure they do not interfere with the performance of the viral assay. It is not uncommon to identify concentrations of sample material that do not cause visible toxicity, but impair the cells or virus enough to prevent the generation of accurate test results. To fully evaluate interference in an assay system, the sample should be tested at multiple dilutions to transect a selection of low virus concentrations (note: the virus concentration will vary depending on the toxicity results and the limits of detection determined by a rigorous assay validation program). The dilution series should cover a wide range of sample concentrations to ensure an endpoint is reached. Reminder: Significant toxic and interfering effects can seriously impact the sensitivity of an assay, and have a detrimental effect on the log10 reduction for a treatment step. Therefore, it is important to conduct the toxicity and interference steps prior to the inactivation / removal portion of the study. Inactivation / Removal Studies
A well-designed manufacturing process is needed to generate high quality product that remains safe and efficacious in the clinic. In addition, critical steps within the purification process must be tested to assess their ability to inactivate virus or physically remove it from the product. Many components of purification processes (e.g., column chromatography, Cohn fractionation) are often very effective at removing specific viruses from the product of interest. Additional treatment steps specifically designed to further remove or inactivate viruses (e.g., viral filters, solvent/detergent treatments) are often included in manufacturing processes. Following are general suggestions about the design of a purification process. | - Where possible, include an inactivation step. Although this is not an absolute requirement, it is good to have a robust inactivation step included in the process. (A description of various inactivation procedures follows later in this section.)
- Filtration can be a useful step to remove virus. Filtration devices often demonstrate reproducible virus removal (applies to multiple purification conditions).
- Search out and avoid overlapping viral clearance steps. When evaluating the performance of the purification process, clearance steps that utilize similar methods of removal or inactivation should be identified. If methods overlap, the log10 reduction values for these steps will not be additive.
Major overlap can be due to a variety of factors. The most common reason for overlap is the failure to remove an inactivating agent before proceeding to the next removal/inactivation step. In addition, many processes possess hidden pitfalls (e.g., columns eluted at a low pH are considered to overlap with a low pH treatment step). As a result, before proceeding with a viral clearance project it is important to discuss the details of the entire process. For further details, please contact your Account Manager.
- As a product moves through the multiple phases of clinical trials, the steps of the process required for viral validation may need to be tested in multiple runs to demonstrate that the log reduction values are consistent each time the process is completed. Also, additional viruses may be required depending on the clinical trial stage and differing international regulatory guidelines. AppTec is experienced with the numerous phases of trials that a drug must go through, from the IND process to the final stages of the BLA. Regulatory submissions are different for each country. These differences can be captured in a custom protocol to meet the needs for specific regulatory submissions.
| Process Steps To Enhance Viral Clearance
Inactivation and Removal Techniques To provide you with general information and useful tips about the various inactivation/removal techniques, following is a summary of the process steps that could be utilized to enhance viral clearance. Inactivation Techniques Many techniques have been demonstrated to inactivate viruses and microbial agents. When selecting an inactivation technique, it is important to first evaluate how the product withstands the treatment step. Another point to remember is that agents used to inactivate virus may also be toxic to the assay system used to measure infectious virus particles, and could reduce the sensitivity of the assay. For example, some viruses (e.g., parvoviruses) are resistant to physical and chemical inactivation. As a result, a treatment step that will inactivate virus could also denature a protein product. When including inactivation techniques in a clearance study there are several factors that should be considered. For example, the concentration of the inactivating agent(s) used in the scaled-down version of the process should be the same as the concentration employed in the full scale manufacturing process, and the process temperature and protein concentrations should remain unchanged. Experience has shown that the protein concentration of the treatment solution can effect the log10 reduction factor (probably due to sequestration of the agent). In addition, when a range of concentrations is used in a treatment, it is advisable to evaluate the worst case scenario (best case for the virus) in the viral clearance study. Finally, when scaling down the process, it is important to evaluate the kinetics of inactivation for the treatment process. This information is necessary to determine if a treatment step is robust. Reminder: When designing a purification process, remove the inactivating agent before proceeding with the next treatment step. Failure to remove the agent will result in overlapping steps.
Following are brief summaries of the treatment steps that are commonly used in manufacturing processes. The summaries contain such information as effective ranges, exposure times and potential pitfalls.
| Heat Treatment
Heat is often used as a terminal treatment step. Effective temperatures depend on the virus to be evaluated in the clearance study. Generally, enveloped viruses are more fragile than non-enveloped viruses and bacteria and, as a result, are inactivated at lower temperatures. For example, at 60° C, most enveloped viruses will be inactivated within 5 minutes. In contrast, robust viruses (e.g., parvovirus and hepatitis A virus) may have to be treated at higher temperatures for a longer exposure time to achieve a comparable log10 reduction value. The effectiveness of the heat treatment can be enhanced if the heating is conducted in solution, rather than in a dry powder format. One pitfall of heat treatment, which is often not considered, is the use of protein stabilizers. Unfortunately, not only do stabilizers protect the product but they often stabilize the spiked virus. This can result in a log10 reduction value that is much lower than expected. Low pH Treatment
Many proteins are able to withstand pH extremes in a range where viruses are inactivated. The effectiveness of low pH treatment of specific viruses is well studied and fairly predictable. In general, enveloped viruses are typically more susceptible to a low pH treatment, or inactivation step. For enveloped viruses (such as retroviruses), the point at which significant inactivation occurs is approximately pH 4.0. At this pH, a short exposure (5 to 30 minutes) is sufficient to inactivate most of the spiked virus. Typically, if the pH is reduced, or the incubation is extended, inactivation by this procedure can be enhanced. Unfortunately, as with most treatments, certain robust viruses (e.g., hepatitis A and parvovirus) are not affected by low pH treatment (even at pH 2.0). As a result, it is advisable to incorporate such a treatment step as one part of a balanced purification process and not to use the step in isolation. One consideration of the Low pH Treatment is its dependence on temperature. When designing this step, particular attention should be paid to the processing temperature of the scale-down model. A low pH step processed at a higher temperature will increase the possibility of inactivating virus, whereas a lower temperature may help to stabilize virus. Solvent / Detergent Treatment
A solvent/detergent treatment is a common addition to many purification processes. This treatment was included as part of most blood product purification techniques to reduce the risk of HIV infection in the 1980s. As a solvent/detergent works by destroying the virus envelope, the treatment is only effective against enveloped viruses and is typically ineffective against non-enveloped viruses. A number of solvents and detergents have been demonstrated to be effective. As with other inactivating treatments, it is important to remove the treatment agent before proceeding to the next purification step. Finally, when designing a process, it is important to remember that solvent/detergent solutions are usually toxic to indicator cells. Ethanol Inactivation
Ethanol treatment is used extensively in the manufacture of tissue products. In addition, ethanol also plays a major part in the safety of products derived from blood due to its involvement in the Cohn fractionation procedure. (See Page I-8 for more information on Cohn fractionation.) Ethanol concentrations known to be effective for virus inactivation vary from virus to virus. Enveloped viruses are generally inactivated relatively quickly (within 5 minutes) by ethanol concentrations greater than 20%. In contrast, prolonged incubation in ethanol greater than 70% is often required to inactivate non-enveloped viruses (such as picornaviruses). Since certain ethanol concentrations are toxic for indicator cells, this factor must be considered in the design of studies. One consideration of Ethanol Inactivation is its dependence on temperature. When designing this step, particular attention should be paid to the processing temperature of the scale-down model. An ethanol step processed at a higher temperature will increase the possibility of inactivating virus, whereas a lower temperature may help to stabilize virus. Irradiation Treatment
Due to the robust nature of many non-enveloped viruses, a number of manufacturers turn to irradiation treatment to enhance their log10 reduction factors. In general, this technique is very effective, although, as with other treatments, the effective dose varies among viruses. This treatment appears to be most effective against viruses with a single stranded genome. Depending on the virus, the reported effective doses range from 25 Kgrays (2.5 Mrad) through to 60 Kgrays (6.0 Mrads). Once again, before including irradiation as part of a process, it is important to test the effect of the treatment on the product. Cleaning Studies
It is necessary for manufacturers to evaluate their cleaning process by evaluating the inactivation of model viruses in the presence of a robust cleaning agent, such as NaOH or HCl. These studies are designed to ensure that there is no cross-contamination between products during manufacturing or purification runs. For cleaning agents being used in manufacturing plants, aliquots of model virus are allowed to dry onto stainless steel surfaces to mimic cleaning processes where product is allowed to dry between manufacturing runs. Cleaning studies can also be performed to evaluate column sanitization solutions / procedures where the column will be regenerated for multiple use chromatography columns. Bacteria, fungi, and mycoplasma are also available to evaluate cleaning studies. Other Treatments
In addition to the treatments detailed above, some less common treatments are also utilized. These include broad spectrum pulsed light, urea, b -propiolactone, hydrogen peroxide, ethylene oxide and protease treatments. For additional information, please contact your Account Manager.
Removal Techniques Column Chromatography
Chromatographic methods can be very effective at removing viruses. When designing this portion of the study, there are many factors that should be taken into consideration. First, it is very important that all the columns to be evaluated during the study are accurately scaled down. During scale down, it is not sufficient to only supply evidence that the elution profiles of the columns are identical. All the column parameters (linear flow rates, residence times, bed height, and HETP values) must be accurately scaled, especially in studies used in support of later stage applications (e.g., BLA). Assuming that the column is accurately scaled, it is necessary to determine which fractions should be collected during the study. It is no longer sufficient to only collect those samples used to determine the log10 reduction value for the column. In addition to a log10 reduction value for a column, it is important to determine which fraction contains the spiked virus. When designing a column clearance step, it is therefore important to conduct a mass balance analysis for the column. If a column is to be reused, it is important to evaluate the capacity of the column sanitization procedure to inactivate virus that may bind to the column. Although the log10 reduction value for the cleaning step is not included in the overall log10 reduction value for the purification process, this is an important supplement for the study. As a product approaches the later phases of clinical trials, column re-use studies also need to be evaluated. The number of column cycles defined for a column in the manufacturing process will determine the life of the resin. It is necessary to examine virus removal for the column resin at the beginning of resin life. End of use resin will then need to be examined to prove that the efficiency of the resin for removing viruses has not diminished over time. Due to the large variety of column resins and differences in the parameters employed by manufacturers, it is not possible to accurately predict the viral clearance values that will be obtained by column chromatography. However, the following list of commonly used columns and the considerations associated with each column provides some insight and general guidelines. - Affinity Chromatography
Affinity columns are an important component of many purification processes. Depending on the virus being evaluated, the clearance factors are generally good with this type of chromatography. In addition, the clearance potential for the column may be enhanced by the fact that a low pH buffer often triggers the elution.
Note: Low pH elution should not be used in combination with a low pH treatment step as these steps will probably be considered overlapping.
- Ion Exchange Chromatography
Ion exchange columns can often produce very good clearance results. However, the results often vary from virus to virus and process to process. Generally, the results for ion exchange chromatography can be optimized if the column is run in an elution mode rather than a flow through mode and the residence time is maximized.
- Other Chromatography Methods
Other separation methods that are used during purification processes include hydrophobic interaction chromatography (HIC), gel filtration and various specialized chromatographic materials. For all of these columns, the log10 reduction values are generally more variable than observed in ion exchange or affinity chromatography. As with ion exchange, a general hint to increase potential clearance for these columns is to elute the column and maximize the residence time of the product on the column. Gel filtration columns generally yield poor viral clearance results.
Filtration
In recent years, specific filter types have become useful tools to enhance the overall viral clearance of many purification processes. In addition, advances in the materials used to construct filters for virus removal (viral filters) have resulted in improved results. Extensive documentation and exceptional technical support makes the scale down of these devices relatively straightforward. Virus filtration works by providing a pore that is able to allow physical separation of the product from the virus particles. Generally, the two types of filters manufactured for this purpose are dead end and tangential flow (TGF) devices. The dead end filter works by passing a fluid through a membrane (or multi-layered structure). In contrast, the tangential flow device works by cycling a fluid reservoir across the surface of a membrane. To determine the log10 reduction value for a viral filter, the starting material is spiked with virus and the mixture is passed through the filtration device. The appropriate samples are collected, the volume is determined and the samples assayed for viral content. As with column chromatography studies, a mass balance should be established for the filtration device. As a result, for the TGF device, both the retentate and permeate samples should be collected and assayed (in contrast to the dead end filter which only generates load and permeate samples). In addition, for dead end filtration, it may be necessary to collect multiple fractions of permeate as the material is processed through the filter. Fractions taken can then be assayed to detect if virus is being held back by the filter throughout the entire filtration or if breakthrough of virus is seen after a certain point in the filtration process. Unlike column chromatography, the expected log10 reduction values for the various filtration devices are very predictable. The choice of device depends on the size of the product, the flow rates, and the protein concentration of the process stream. Generally, the devices will remove viruses greater than
50 nm in size. In addition, some devices have the potential to remove even the smallest virus particles. Cohn Fractionation
The Cohn fractionation process has been used extensively in the blood industry to purify products from human plasma. The process works by adjusting ethanol and temperature conditions to generate a controlled partitioning process. Historically, this technique has proven to be a very good safety barrier against viral contamination. As detailed above, good log10 reduction factors often obtained with this method (particularly for enveloped viruses) are probably due to a removal technique used in combination with high ethanol concentrations (often in excess of 25%), which inactivate virus. Although the technique has a very good safety record, there are some concerns about this process and its use in scale-down clearance studies. First, the process is difficult to accurately scale down. As a result, data supporting the scale down protocol must be included in the license application. Furthermore, due to the difficulties in scale down, it is recommended that the process be replicated in the clearance study to demonstrate that the process is reproducible. In addition to the issues concerning the scale down, manufacturers are often required to determine, when possible, if the process removes or inactivates the spiked virus. As the Cohn fractionation probably works by combining inactivation and removal techniques, this detailed breakdown is often not possible. | | : | | Study Enhancements Back to Top | Real Time PCR
Samples for various viral removal or inactivation steps can be quantitatively assessed for both infectivity and total genome copies. Classic viral infectivity studies can only detect the amount of infectious virus present in a sample. Incorporating real time PCR (qPCR) into a viral clearance study allows the viral genomic copy number to be deterimined. This allows purification steps to distinguish between true virus removal and inactivation.
Please refer to the Molecular Biology section of this catalog for information on qPCR assays.
Large Volume Testing
A larger sample volume can be tested for those samples that are expected to yield non-detectable infectious virus. If a larger volume is tested, this increases the sensitivity of the assay. Therefore, a greater log reduction value (LRV) can be claimed. If a process contains one robust step, a larger LRV may be critical to satisfy safety guidelines. Reduction of Toxicity on Indicator Cell Lines
In an attempt to inactivate viruses, clients often use chemicals that generate reactive oxygen species. One of the most commonly used of these chemicals is hydrogen peroxide (H2O2). While this chemical is very efficient at inactivating viruses, it suffers the drawback of being highly toxic to many cell lines used to assay for virus. This necessitates large dilutions of the samples for testing, thus reducing the sensitivity of the virus inactivation assay. We have attempted to reduce the toxicity of solutions containing reactive oxygen species by assaying for virus using a new proprietary media supplement. The reductions in toxicity using the modified media allow for a much more sensitive detection of virus in samples containing reactive oxygen species. This increase in ability to detect virus increases our maximum theoretical clearance values. Assuming complete viral inactivation by the treatment, the log reduction values can provide an increase in our ability to detect virus by as much as 4.5 logs. | | : | | Virus / Organism Selection Back to Top | | One of the most important components in the design of a viral clearance study is the selection of viruses to be evaluated. The selection of model viruses is based on the source of the material from which the product is derived and on the characteristics of potential adventitious viral contaminants. In addition, the virus panel should represent a wide range of physical and chemical properties. An ICH document ("Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnological Products Derived From Cell Lines of Human or Animal Origin") separates viruses into three main categories: relevant viruses, specific model viruses and non-specific model viruses. Relevant Viruses
Relevant viruses are viruses that are either present in the starting material (e.g., MuLV in murine cell lines), or could potentially contaminate the cell substrate or starting material (e.g., HIV in human blood products). Specific Model Viruses
When a relevant virus is not available, or cannot be grown to a sufficiently high titer in vitro, a specific model virus can be used. Model viruses are often very closely related to viruses of concern (e.g., bovine viral diarrhea virus is a specific model virus for hepatitis C virus and duck hepatitis B virus is a specific model virus for human hepatitis B virus) and are presumed to demonstrate a similar log10 reduction value. AppTec offers specialized testing of FDA- and EPA-regulated products in a duck hepatitis B virus (DHBV) model to measure the removal or inactivation of hepatitis B virus. Non-specific Model Viruses
To demonstrate that a purification step is robust, it is important to have a well rounded virus panel, including specific model viruses as well as non-specific model viruses. Non-specific model viruses are included to show clearance of infectious viral particles (known or unknown) that could enter the process undetected. This requires that a virus from each of the major categories (enveloped, DNA; non-enveloped, DNA; enveloped, RNA; and non-enveloped, RNA) be included. | | : | | Virus Selection Back to Top |
Viruses Offered for Clearance Studies
Virus | Virus Family | Envelope | Genome | Approx. Size (nm) | Adenovirus type 5 | Adeno | No | DNA | 70 - 90 | Amphotropic Murine Leukemia Virus | Retro | Yes | RNA | 80 - 130 | Bovine Parainfluenza Virus | Paramyxo | Yes | RNA | 150 - 300 | Bovine Parvovirus | Parvo | No | DNA | 18 - 26 | Bovine Viral Diarrhea Virus | Flavi | Yes | RNA | 60 - 70 | Cytomegalovirus | Herpes | Yes | DNA | 180 - 200 | Duck Hepatitis B Virus | Hepadna | Yes | DNA | ~40 | Encephalomyocarditis Virus | Picorna | No | RNA | 28 - 30 | Hepatitis A Virus | Picorna | No | RNA | 28 - 30 | Human Immunodeficiency Virus | Retro | Yes | RNA | 80 - 130 | Herpes Simplex Virus, Type 1 | Herpes | Yes | DNA | 150 - 200 | Infectious Bovine Rhinotracheitis Virus | Herpes | Yes | DNA | 150 - 200 | Influenza A Virus | Orthomyxo | Yes | RNA | 80-120 | Minute Virus of Mice | Parvo | No | DNA | 18 - 22 | Poliovirus | Picorna | No | RNA | 28 - 30 | Porcine Parvovirus | Parvo | No | DNA | 18 - 26 | Pseudorabies Virus | Herpes | Yes | DNA | 150 - 200 | Reovirus type 3 | Reo | No | RNA | 60 - 80 | Simian Virus 40 | Papova | No | DNA | ~45 | Transmissible Gastroenteritis Virus | Corona | Yes | RNA | 80 - 160 | Vesicular Stomatitis Virus | Rhabdo | Yes | RNA | 75 x 180 | Xenotropic Murine Leukemia Virus | Retro | Yes | RNA | 80 - 130 |
| | : | | Quality Program for Virus Stocks Back to Top | | As virus stock solutions used in clearance studies are governed as Control Articles under Section 58.105 of the GLP (Test and Control Article Characterization), it is important that these solutions be tested for identity, strength and purity. To meet these requirements, AppTec has established new criteria to obtain the highest quality virus stocks in the industry. The program includes testing of master stock, working stock and production lots to ensure the quality of the virus stocks. Virus stock preparations begin with the acquisition of virus from only the most reputable sources. To ensure purity, all viruses are routinely triple-plaque purified (or triple purified by end point dilution) in host cell lines that are derived from AppTec’s cell bank. It is important to note that an analogous system is in place for the generation of fully tested, certified cell banks. All virus testing and production is conducted using cells derived from these cell banks. Individual virus clones/plaques are carefully selected as seed stocks of pure, infectious virus. These seed stocks are subsequently used to generate master virus seed stocks that have undergone limited rounds of virus replication. The master virus seed stock is fully tested for identity, purity and potency. Identity testing includes the use of multiple immunologic, viral and molecular biological techniques to confirm the identity of the reagent. Purity is determined by testing for a wide variety of potential viral and microbiological contaminants. Potency is determined using fully validated specific viral assays to accurately titrate the stock solution. Working virus seed stocks are generated from the master virus seed stocks in appropriate host cell lines. Once completed, representative vials of the working virus seed stocks are fully tested (as outlined above) to confirm the identity, purity and potency of this intermediate virus stock. Finally, high titer production lots of virus are generated from the working virus seed stock for use in viral clearance studies. These stocks are fully tested a third time to insure that no adventitious agents were introduced during the process. Overall, we believe this multi-tiered system for virus production and characterization assures the highest quality of virus. AppTec is pleased to be able to offer these virus stocks for use in your viral clearance studies. Also available are column-purified virus stocks for custom testing where pure viral preparations may be required or where appropriate. | | : | | Virus Assay Validation Back to Top | | In recent years, the science and technology being used to conduct viral clearance studies has become increasingly sophisticated and rigorous. One requirement in this continually evolving field is that the virus titration assays used to detect virus must be thoroughly validated. To address these changes, AppTec developed a thorough assay validation program to ensure that all of the viral assays currently offered for viral clearance studies meet the highest standards. When designing an assay validation program, guidance can be obtained from a selection of documents. These include Notes for Guidance on Validation of Analytical Methods: Definitions and Terminology issued by the International Committee on Harmonisation (ICH) and the Note for Guidance on Virus Validation Studies: The Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses issued by the Committee for Proprietary Medicinal Products (CPMP). Performance Characteristics Viral assays share some characteristics with tests designed to quantitate the major constituents as well as the impurities in products. Validations of this type are defined in the USP XXIII, Section 1225, Validation of Compendial Methods and in the Notes for Guidance on Validation of Analytical Methods: Definitions and Terminology issued by the ICH. These regulatory documents state that assay validation should quantitate the following performance characteristics: Precision (Including repeatability, intermediate precision, and reproducibility)
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple samplings of the same homogeneous sample under the prescribed conditions. Precision may be considered at three levels: repeatability, intermediate precision, and reproducibility. Repeatability (intra-assay precision) expresses the precision under the same operating conditions over a short interval of time. Intermediate precision expresses within-laboratory variations (different days, different analysts). Reproducibility expresses the precision between laboratories. Since all samples generated during a viral clearance study are assayed in the same laboratory, this parameter is not relevant. The CPMP and ICH guidelines for viral safety evaluation of biotechnology products require that the 95% confidence limits on calculated titers for within-assay variation and between-assay variation be in the order of ± 0.5 log10 of the mean. Note: The regulatory documents for viral clearance studies currently require a 95% confidence limit rather than a standard deviation as a measure of precision. Use of the standard deviation can provide an inaccurate indication of assay precision. Accuracy
The accuracy of an analytical procedure expresses the closeness of agreement between the true value, a conventional true value, or a reference value and the mean of the results obtained by applying the analytical procedure a number of times. Given that the infectivity titer of a virus preparation differs depending on the virus strain used, the indicator cell line used and the experimental conditions used, a true value cannot be established. To determine accuracy, a conventional true value is established by repeated titration of a virus stock solution in that assay system. Titers determined in additional titrations are then evaluated relative to that conventional true value. Note: For viral clearance studies, the reduction factors are calculated as an estimate of relative titers. Given this, high precision is the essential factor for an assay used in this application. Limit of Quantitation
The limit of quantitation is the lowest concentration of analyte in a sample that can be quantitated reliably with accuracy and precision. For viral infectivity assays, when virus concentrations are low and the volume tested is small relative to the total sample volume, the regulatory authorities recommend that an estimate of the limit of quantitation be obtained using Poisson distribution. In addition to the Limit of Quantitation, a Limit of Detection can also be determined. This is the lowest amount of an analyte that can be detected but not necessarily quantitated with accuracy and precision. Limit of detection is not a performance characteristic for quantitative assays according to the USP and ICH documents on assay validation. Note: The limit of detection should not be substituted for the limit of quantitation, as this value cannot be quantitated with accuracy and precision. Linearity and Range
The linearity of an analytical procedure is its ability, within a given range, to yield test results that are proportional directly or through mathematical transformation to the concentration of analyte in the sample. The range of an analytical method is the interval between the upper and lower concentration of analyte in the sample, including these concentrations, for which the assay has demonstrated a suitable level of precision, accuracy and linearity. The linearity of an assay may be calculated by the least squares method and represented as the Coefficient of Determination (r 2). Note: It is important to clearly differentiate between the coefficient of correlation (r) and the coefficient of determination (r 2). Using r instead of r 2 implies a better fit of the data than actually exists. Specificity
The specificity of an analytical method is its ability to reliably quantitate the analyte in the presence of compounds that may be expected to be in the sample. Given that the nature of the samples analyzed at AppTec varies considerably with respect to species, tissues source, purity, and concentration, it is not possible to address all potential interfering conditions/substances in a general validation study. AppTec addresses the issue of specificity in a client product-specific manner by performing toxicity and interference studies prior to the initiation of viral clearance studies. | | : | Regulatory Documents Back to Top | When designing viral clearance studies the following regulatory documents should be consulted.
U.S. Food and Drug Administration (FDA) Center for Biologics Evaluation and Research (CBER). 1997. "Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use." 94D-0259
U.S. Food and Drug Administration (FDA) Center for Biologics Evaluation and Research (CBER). 1993. "Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals."
Committee for Proprietary Medicinal Products (CPMP). 1997. "International Conference on Harmonization (ICH) Topic Q 5 A. Quality of Biotechnological Products: Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin." Consensus Guideline ICH Viral Safety Document: Step 4. CPMP/ICH/295/95
Committee for Proprietary Medicinal Products (CPMP). 1996. "The Design, Contribution and Interpretation of Studies Validating the Inactivation and Removal of Viruses." CPMP/BWP/268/95 | Back to Top |
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