Introduction
Viral contamination events have occurred in a number of production processes over the last 20 years. In most cases, the source was either proven or suspected to be a raw material component. Most of the contamination events have occurred with non-enveloped viruses, which lack an outer lipid envelope. Testing for virus in raw materials, starting materials, or in the unprocessed bulk product can assist in ensuring that the product will be free of detectable viral contamination. However, some viruses may not be detected in the assays, and therefore the risk of virus contamination cannot be fully controlled by testing alone.
Viral Clearance Studies
Viral clearance studies play a crucial role in assuring that biopharmaceutical products made in animal cells are free of virus that could potentially contaminate the product. In viral clearance studies, selected steps in the product’s purification process are deliberately spiked with selected viruses, and the ability of the step to remove and/or inactivate the virus is evaluated. A clearance factor for the entire process can be calculated for each virus by adding results from all of the steps tested. Results of viral clearance studies demonstrate that undetected viruses that may contaminate the unprocessed bulk can be effectively cleared from the product during the purification process.
Mechanisms of Viral Clearance
Purification process steps can act in two different ways: by separating the virus from the product stream (termed removal steps), and by inactivating the virus so that it is no longer infectious (inactivation steps). Some steps may act in both ways. Inactivation steps, such as low pH, detergent, or irradiation, are important contributors to the total clearance obtained, and regulatory agencies expect to see at least one effective inactivation step in a purification process. However, nonenveloped viruses are significantly more difficult to inactivate than enveloped viruses. Of particular interest is Murine Minute Virus (MMV), which has been detected as a viral contaminant of CHO cells. Most purification processes have no inactivation step that is effective for MMV.
Viral Inactivation Using UV-C Irradiation
Although UV-C irradiation has been used for water treatment for a number of years, its use with biopharmaceutical products is fairly new, and there is limited information available on its effectiveness in inactivating viruses. UV-C irradiation is performed with monochromatic light at 254 nm, which targets nucleic acids preferentially over proteins. We have evaluated this technology for its effectiveness with viruses from seven different families (Table 1). The instrument used in these studies was a GMP-compliant lab scale UV-C irradiation system. In this instrument, the solution is pumped through a helical reactor coil that surrounds the UV bulb. The UV dosage is controlled by the light intensity and the flow rate.
Table 1. Viruses Used in the UV-C Inactivation Study.
Each virus was spiked into a model protein solution and then irradiated at several different doses. Results from the study are shown in Figure 1. It can be seen that MMV and PPV are the most sensitive to UV-C radiation of all the viruses evaluated. The other viruses showed a spectrum of inactivation, with xMulV being the least sensitive. It can be seen from the results that the relative sensitivity of viruses to UV-C irradiation is not correlated with virus size; presence or lack of a lipid envelope; or genome type, size, or strandedness. Since the mechanism of action of UV-C is by formation of pyrimidine dimers, it is likely that the sensitivity to inactivation is related to the frequency of sequential pyrimidines in the genome.
Figure 1 - Inactivation of Diff erent Viruses by UV-C IrradiationWe performed additional experiments to evaluate the effects of various process conditions on inactivation. There was no significant difference in inactivation at protein concentrations from 5 to 30 mg/ml. There was also no significant difference in inactivation in acetate buffer ph 5.5, PBS ph 7.2, or Tris buffer ph 8.5. Because UV inactivation is insensitive to variations in these parameters, it is a very versatile viral inactivation step that can be used in many places in a purification process.
Applications and Considerations
UV-C irradiation may be included in the purification process as a dedicated step for inactivation of viruses. UV-C irradiation may also be used as a barrier treatment of raw materials before they are added to the bioreactor. Since raw materials are in most cases not affected by UV-C irradiation, higher doses can be used for this application, resulting in more efficient inactivation.
There are a few factors to keep in mind when considering whether to include UV-C in a purification process.
- UV-C is highly effective in inactivation of MMV. however, it is not effective with all viruses, particularly retroviruses, so it cannot be used as the only inactivation step in a process.
- Although nucleic acids absorb the radiation preferentially, proteins do absorb radiation to some extent. Therefore, UV-C radiation may cause product damage. Product damage, including aggregation or changes in immunogenicity, has been observed, but at moderate doses (200-300 J/m2) effective inactivation of viruses can be obtained while limiting the protein damage. It is a good idea to place the UV-C step upstream of a polishing step, so that any product that is damaged can be removed.
- The intensity of irradiation available for inactivation is inversely related to the absorbance of the solution at 254 nm. If the process intermediate contains a high level of nucleic acids or is very concentrated, it may be difficult to obtain a dose high enough to be effective. Conversely, if the product is pure and at low concentration, the minimum dose that can be obtained may be too high and product damage could result. These factors must be considered when determining where this step should be placed in a purification process.
Conclusion
In summary, UV-C irradiation is a highly versatile viral inactivation step that can be included in a purification process in a number of ways. Since it is a flow-through operation, adding it to a process is relatively simple. The procedure is highly effective with parvoviruses and is therefore a useful addition to the repertoire of process steps for viral clearance. UV-C irradiation can also be an effective barrier step for treatment of raw materials.
Katherine Bergmann, PhD, is manager of Viral Safety and Viral Clearance Services at Eurofins Lancaster Laboratories. She has extensive experience in cell line characterization, lot release testing and viral/TSE clearance validation and has numerous publications and presentations in the field. She has performed biomedical research in protein chemistry, cell biology and virology at Georgetown University, Scripps Research Institute and Rockefeller University. She received a PhD from Brown University and a AB degree from Mount Holyoke College. Dr. Bergmann served on the PDA task force on Preparation of Virus Spikes Used for Virus Clearance Studies.