Initial Evaluation
for Virus Safety 

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Step 2

Once you have completed the Assessment of Needs, it is time to take a look at the protein purification process, at both your current scale and at future manufacturing scale. The goal is to ensure virus filtration is implemented in an optimal and robust way.

Virus Filtration Parvoviruses have a diameter of ~18-26 nm, but a typical monoclonal IgG antibody has a hydrodynamic diameter of ~8-12 nm. To achieve >4 log10 retention of the viruses and a >99% recovery of the protein, parvovirus filters are required to have a small nominal pore size and very narrow pore size distribution and are, therefore, sensitive to the presence of impurities in the feed solution.

The sensitivity to impurities and relatively small size of the virus and monoclonal antibody can be overcome via prefiltration and the design and placement of the downstream purification step. Merck has 20 years of experience in assisting our customers in the design and implementation of Virus Clearance operations - we can help.

Several process parameters have a significant impact on the downstream process train, such as:

1. Location:

Typically, a normal flow virus filtration step can be implemented at any one of several points in a given downstream process. For example, in a typical monoclonal antibody process, the filtration can occur in any one of three locations in the downstream purification process.

Normal flow virus filtration step: - Following the low pH inactivation step - Following an intermediate column chromatography step - After the final column chromatography step

2. Feed Concentration:

Feed solution concentration can affect the virus filtration process by reducing product throughput. The level of impact will depend on the interaction between the filter and the components in the solution being filtered.

Feed solution concentration: The effect of protein concentration on the filter area needed can be significant. In this case, the optimum concentration is between 8 and 10 g/L. The optimum concentration can vary, depending upon the protein purity and the buffer conditions.

3. Prefiltration:

Prefiltration of the feed solution can have a dramatic impact on filter performance. Prefiltration is targeted to remove various impurities and contaminants such as protein aggregates, DNA, and other trace materials.

4. Hold Times and Freeze-Thaw Cycles:

Some proteins exhibit time-dependent aggregate formation or will form low concentrations of aggregates when subjected to a freeze-thaw cycle. If a hold step or a freeze-thaw cycle is expected to be a part of the validation process, it is important to evaluate the effects of this during filter optimization.

5. Process Time:

Parvovirus filters can be broadly classified into two groups – those with high protein flux but low-to-moderate volumetric capacities, and those with high protein capacities but with low-to-moderate protein fluxes. Both of these filters have advantages and disadvantages. When a process must be completed in a short amount of time (2-4 hours), high flux filters require less filtration area than high capacity filters. On the other hand, when processing times are extended to 6 hours or more, high-capacity filters may be more economical.

Advantages and disadvantages of parvovirus filters: The graph shows the filtration area in m2needed to filter 1,000 L of protein as a function of the process time through different parvovirus filters. For short processing times (2-4 hours), high flux filters generally require less filter time that high capacity filters.

During this phase Merck can assist you in designing and running your experiments, at your lab or our facility. We have created a proprietary sizing methodology so that you can select the smallest filter able to give you optimal throughput.

Contact the Viresolve® Team for more information.