Techniques for Viral Inactivation
Testing and screening of cell substrates and raw materials represents one part of the approach to demonstrate viral safety.
The testing strategy will only detect viruses targeted in the assays, and there is the possibility that unknown viruses will
remain, for which the assays are not designed to detect. It is, therefore, important to incorporate specific steps into the
purification process that will remove or inactivate viruses with different physicochemical properties.
Products such as monoclonal antibodies (mAbs) are typically manufactured using a generic platform type of process. Two key
steps are incorporated into the purification of a mAb to prevent viral contamination: incubation at low pH to inactivate enveloped
viruses and nanofiltration to remove both enveloped and non-enveloped viruses. The regulatory authorities require at least
two robust viral-reduction steps within the process to ensure that sufficiently low levels of contamination can be achieved
to ensure patient safety. These methods must demonstrate the ability to remove viruses by different mechanisms, for example,
a removal step and an inactivation step.
Low pH treatment is an inactivation-based technique. It can be simple to introduce into the process, particularly when the
mAb is purified using a Protein A-based affinity step as the first capture step when the bound product is eluted by reducing
the pH of the buffer. The process provides a logical point in the process to implement a low pH viral-inactivation treatment
step. The pH of the sample is adjusted to a specific pH range, typically between 3.5 and 3.9. It is held at that pH for a
predefined period of time, typically one to four hours. This step has been shown to be effective at inactivating a number
of enveloped viruses. However, in general, it only inactivates viruses that are categorized as having a low, or low-to-medium,
resistance to physicochemical influences. Manufacturers typically use spiked samples of model viruses such as murine leukaemia
retrovirus (MLV) and pseudorabies herpesvirus (PRV) to evaluate the effectiveness of this step (see Table I). When a step such as this is incorporated into product purification, it is important to ensure that the product itself will
be stable at these process conditions. Protein products can, occasionally, be susceptible to acidic cleavage, and thus not
particularly stable when kept at low pH for long periods. If this is the case, then alternative viral inactivation methods
should be considered.
Table I: Examples for 60-minute hold time.
Alternative methods include solvent-detergent treatment, which interferes with the lipid coat of the virus. In this treatment,
the product is incubated in a defined concentration of solvent (typically 0.15 to 0.3% TnBP), which accelerates the interaction
between that lipid coat and a detergent—usually Triton X-100 or Tween 80 (typically 0.1 to 1%). The disruption caused by the
solvent and detergent to the viral membrane inactivates the virus. Again, this method is particularly suitable for enveloped
viruses. This technique is commonly used for plasma-derived products, having originally been developed for use in the blood-products
Other techniques for viral inactivation include gamma irradiation, which is commonly used to sterilize finished medical devices.
UV irradiation is slowly becoming more common for mAbs. Formaldehyde treatment is also used occasionally, while treatment
with beta-propiolactone is routine for vaccine preparations. Raw materials, particularly those used upstream in the process,
may be treated to inactivate potential pathogens using gamma irradiation, UV irradiation and high-temperature, short-time
(HTST) pasteurization. Not all inactivation methods are suitable for use on the raw materials and the potential impact on
functionality should be assessed before implementing into the process.
Non-enveloped viruses are particularly resistant to inactivation and are typically unaffected by low pH and resistant to solvent
detergent treatment. Other methods of inactivation may need to be considered for these viruses.