VIRAL INACTIVATION
Virus
Murine Minute Virus (MMV) Porcine Parvovirus (PPV) Polio Virus
Bovine Viral Diarrhea Virus (BVDV) Reovirus type 3 (Reo 3) Adenovirus type 5 (Ad 5)
Xenotropic Murine Leukemia Virus (XMuLV) Pseudorabies Virus (PRV)
Family Parvo Parvo
Picorna Flavi Reo
Adeno Retro
Herpes
Table 1. Viruses Used in the UV-C Inactivation Study. Size (nm)
18-24 18-24 25-32 50-70 60-80
90-100 80-110
120-200
Genome DNA DNA RNA RNA RNA DNA RNA DNA
Envelope No No No Yes No No Yes Yes
Strand SS SS SS SS DS DS SS DS
Genome size (kb) 4-6 4-6 7-8
10-11 18-30 26-46 10-11
150-200 Figure 1 - Inactivation of Diff erent Viruses by UV-C Irradiation
• 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
)
eff ective 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.
Each virus was spiked into a model protein solution and then irradiated at several diff erent 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.
We performed additional experiments to evaluate the eff ects various process conditions on inactivation. There was no signifi cant diff erence in inactivation at protein concentrations from 5 to 30 mg/mL. There was also no signifi cant diff erence in inactivation in acetate buff er pH 5.5, PBS pH 7.2, or Tris buff er 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 purifi cation process.
Applications and Considerations
UV-C irradiation may be included in the purifi cation 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 aff ected by UV-C irradiation, higher doses can be used for this application, resulting in more effi cient inactivation.
There are a few factors to keep in mind when considering whether to include UV-C in a purifi cation process.
• UV-C is highly eff ective in inactivation of MMV. However, it is not eff ective with all viruses, particularly retroviruses, so it cannot be used as the only inactivation step in a process.
13 American Pharmaceutical Review | Biopharmaceutical Supplement 2014
• 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 diffi cult to obtain a dose high enough to be eff ective. 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 purifi cation process.
Conclusion
In summary, UV-C irradiation is a highly versatile viral inactivation step that can be included in a purifi cation process in a number of ways. Since it is a fl ow-through operation, adding it to a process is relatively simple. The procedure is highly eff ective with parvoviruses and is therefore a useful addition to the repertoire of process steps for viral clearance. UV-C irradiation can also be an eff ective barrier step for treatment of raw materials.
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