At Charles River, we understand how critical it is to demonstrate that the manufacturing process for your biologic appropriately removes or inactivates any known contaminants. Manufacturers of biopharmaceutical products, products derived from animal or human tissues, blood products, and some medical devices are required to assess the ability of their purification and manufacturing processes to produce a product that is safe for use in humans. Studies designed to demonstrate the capacity of the downstream process to inactivate or remove viruses and transmissible spongiform encephalopathy (TSE) agents are required by regulatory authorities as an integral part of your submission report prior to administration of a product in humans.
Our scientists at Charles River Biopharmaceutical Services (BPS), located in both the US and Europe, have more than 20 years of experience in supporting clients throughout the design and performance of viral and TSE clearance studies. Using a customized approach, we can provide technical advice and regulatory support to ensure that a successful program is established and reported to meet your deadlines. Our studies are performed under strict regulatory compliance according to Q5A, FDA Points to Consider, and the EMEA Guidelines of Viral Safety Evaluation.
Product Experience
• Monoclonal antibodies
• Recombinant proteins
• Transgenic products
• Tissue-derived products
• Blood-derived products
• Medical devices
Three complementary approaches are used to ensure the viral and TSE safety of a biotechnology product. First, source cell lines and raw materials are tested for freedom from viruses. Second, in-process testing programs are used to assess the presence of virus and TSE in the crude product and to ensure their absence in the final product. The third component is the assessment of viral clearance during the purification process.
Clearance is a measure of the capacity of the purification process to primarily inactivate viruses and prions. These studies involve deliberate spiking of virus or TSE agents into process intermediates and then demonstrating their inactivation or removal during the subsequent processing steps.
Before performing a viral or TSE safety study, a thorough analysis of the potential for viral contamination of the manufacturing process should be conducted. This involves research on the history of the cell substrate, characterization of master and working cell banks and end-of-production cells, raw material testing, and bulk harvest testing. Analysis of the bulk harvest material by electron microscopy (EM) is performed to obtain a measure of the viral load prior to purification.
The scope and design of a viral or TSE safety study involves:
• Selection of the process steps
• Verification of the scaled-down process steps
• Viruses or TSE spike solution selection
• Selection of viruses or TSE spike solutions
• Determination of test article cytotoxicity (virus) or compatibility (TSE)
• Performance of the process steps in the presence of virus/TSE spike
• Evaluation of the virus/TSE removal and inactivation
Selection of Process Steps
It is not necessary to test all steps in a purification process if sufficient clearance can be shown by analysis of fewer steps. Process steps should be selected where there is a reasonable expectation of viral clearance. Additional factors for consideration are the ease of scale-down of the procedure and the reproducibility of the procedure under process conditions. Results from such steps will provide greater confidence that the results of the viral clearance studies will translate to the full-scale manufacturing process.
Verification of the Scaled-Down Process
With any process validation study, it is critical to establish the validity of the scaled-down version of the purification steps. The scaled-down process should also accurately reflect the actual manufacturing process prior to the implementation of the study. This test process is then evaluated in a biosafety laboratory for its capacity to inactivate or remove viruses/TSE. The validity of the scaled-down process should be demonstrated by comparing process parameters for similarity with the manufacturing-scale process and by verifying that the authenticity and stability of the process intermediates used in the study are the same as those produced in the manufacturing process. Process parameters should be selected to represent specific operating conditions and acceptable tolerances to the process of the manufacturing scale. Our considerable in-process experience (over one thousand studies) allows us to partner with you in the development and validation of the scaled-down versions of the steps, as well as supply much of the standard equipment that is commonly used in protein purification, such as the equipment needed for inactivation studies and nanofiltration, chromatography devices, and lyophilizers.
Selection of Viruses or TSE Spike Material Selections
Viruses that are intended for use in viral clearance studies should be model viruses that contaminate or may contaminate the product. The model viruses should also present a wide range of physiochemical properties in order to evaluate the robustness of the purification process to remove or inactivate a range of viruses. The effect of the sample on the virus replication must be determined by comparing the virus titer from titration experiments performed in the presence of sample to those performed in the absence of the sample.
Viruses Available for Viral Clearance
|
Type |
Viruses |
Family |
Size (nm) |
|
RNA enveloped |
|
|
Human immunodeficiency virus-1 |
Retrovirus |
80 - 110 |
|
|
Xenotropic, amphotropic,
and ecotropic recombinant
murine retroviruses |
Retrovirus |
80 - 110 |
|
|
Bovine viral diarrhea virus |
Flavivirus |
40 - 60 |
|
|
Vesicular stomatitis virus |
Rhabdovirus |
70 - 150 |
|
|
Visna virus |
Retrovirus |
80 - 110 |
|
|
Parainfluenza type 3 |
Paramyxovirus |
100 - 200 |
|
RNA non-enveloped |
|
|
Reovirus type 3 |
Reovirus |
60 - 80 |
|
|
Poliovirus type 1 |
Picornavirus |
23 - 30 |
|
|
EMC virus |
Picornavirus |
25 - 30 |
|
|
Hepatitis A virus |
Picornavirus |
25 - 30 |
|
DNA enveloped |
|
|
Pseudorabies virus |
Herpesvirus |
120 - 200 |
|
|
Herpes simplex virus type 1 |
Herpesvirus |
120 - 200 |
|
|
Orf virus |
Poxvirus |
140 - 450 |
|
|
Infectious bovine rhinotracheitis virus |
Herpesvirus |
120-200 |
|
DNA non-enveloped |
|
|
Human adenovirus |
Adenovirus |
70 - 90 |
|
|
Simian virus 40 |
Papovavirus |
40 - 50 |
|
|
Porcine parvovirus |
Parvovirus |
18 -24 |
|
|
Murine minute virus |
Parvovirus |
18 -24 |
Depending on the process step to be investigated, a variety of TSE preparations are available for spiking experiments. The physicochemical nature of TSE agents in blood or other body fluids is still unknown, but they are assumed to be present in low concentrations in a soluble monomeric or oligomeric form.
Determination of Test Article Cytotoxicity or Compatibility
For viral clearance studies, a cytotoxicity test must be performed to determine if and to what degree the test item must be diluted so that it has no toxic effects on the indicator cells. After incubation with the process samples, the indicator cell lines are inspected microscopically for cytopathic effects including changes in cell morphology, loss of cell adherence, syncytia formation, and cell death.
For a TSE program, each test article matrix must be tested for its compatibility with the conditions of gel electrophoresis and western blotting to ensure valid results. These two techniques are critical to identifying key steps in TSE removal or inactivation.
Determination of the Existence of Viral/TSE Interference
The effect of the sample on the virus replication must be determined by comparing the virus titer from titration experiments performed in the presence or absence of the sample.
Performance of the Process Steps in the Presence of Virus or TSE Spike
Virus is spiked at high titers into the sample prior to performing the scaled-down purification method. The titer of the virus in the stock solution and of the samples taken during the process itself is determined by end-point titration.
The common principle of a TSE safety study is to define the removal or inactivation capacity for TSE particles in individual process steps. This is done by spiking the relevant process intermediates with infectious TSE particles, followed by the determination of residual TSE particles after the defined process steps.
Evaluation of Virus/TSE Removal or Inactivation
A TCID50 assay or plaque assay can be used to determine the viral titer of the samples. The actual number of samples that should be analyzed depends on the process step itself and must be individually defined within each viral safety study. Alternative methods such as quantitative PCR (Q-PCR) may be employed to understand the mechanism of viral clearance in more detail.
To identify key steps of TSE removal or inactivation, the samples are analyzed using a western blot to detect the prion protein in its abnormal confirmation.
To identify key steps of virus removal or inactivation, we utilize the logarithmic reduction factor for each individual process step and virus. The actual number of samples that should be analyzed depends on the process step itself and must be individually defined within each viral safety study. Alternative methods such as Q-PCR may be used to understand the mechanism of viral clearance in more detail. For TSE removal or inactivation, the samples are analyzed using Western blot.
Summary
Viral and TSE safety studies are an essential part of a manufacturer’s program to ensure product safety. In conjunction with characterization of starting materials and a program of bulk and final product testing, a careful analysis of the capability of the manufacturing process to remove or inactivate a wide range of test viruses or TSE agents plays an important role in establishing the safety of biological products.