Choice of steps
Prior to launching any experiments, a detailed analysis of the manufacturing process has to be done in order to identify the steps that are theoretically able to eliminate and/or inactivate viruses. Thanks to 19 years of virus validation experience, we help you choose the most efficient viral inactivation treatments and the optimal virus elimination steps, depending on your product.
In terms of virus clearance, we distinguish three different kinds of steps for evaluation:
- inactivation steps
- removal steps
- both inactivation and removal steps
Inactivation steps
The most common inactivation treatments are:
- heat treatment (pasteurisation, autoclaving, etc.),
- chemical treatment (acid or alkaline pH, H2O2, solvents, detergents, etc.),
- physical treatment (high pressure, UV, gamma rays, supercritical treatment etc.),
- enzymatic digestion
As mentioned into the official guidelines, it is of paramount importance to discover the cause or causes of viral inactivation. The observation of viral reduction is itself not sufficient; it is also necessary to demonstrate that the virus has been inactivated by a specific parameter. If several parameters are involved simultaneously, it is necessary to analyse the contribution of each. This requires a large number of controls and, in particular, an approach which consists in "neutralizing" one potentially inactivating parameter with respect to manufacturing process conditions.
When a match has been found between the theoretical analysis and the experimental observation for a major viral inactivation parameter, it is necessary to plot an inactivation curve as a function of time. Where the inactivation is too rapid to plot the kinetics using process conditions, we suggest either using additional controls where the inactivating parameter is neutralised or plotting the kinetics data more accurately by "minimizing" the inactivating parameter. This approach will also provide interesting information on the robustness of the step.
Removal steps
The most common removal steps are:
- nanofiltration.
- chromatography (Q-sepharose, SP sepharose, protein A columns, etc.),
- precipitation.
When a virus partition occurs during a production step under evaluation, it is necessary to determine the viral load in each "fraction" generated and thus to demonstrate the viral balance. For example, a viral balance should be calculated for steps like protein precipitations or chromatography operations. In this latter case, the gel itself represents a fraction that should be analysed.
Both inactivation and removal steps
Special attention should be paid when evaluating steps that provoke simultaneous elimination and inactivation (i.e. protein A chromatography with elution at acidic pH). In fact, it is not permitted to evaluate the same inactivating factor twice in a purification process. Hence, it is important to distinguish between viral elimination and viral removal. To this end, we double- titrate samples using a quantitative PCR (qPCR) test that solely detects viral genomes, even if the viruses themselves have been inactivated. The combined interpretation of infectious titres and qPCR results enables us to distinguish between virus removal and inactivation and thus to obtain the best possible LRV.
