What is Nanofiltration?

Nanofiltration is a physical method used to remove viruses from biological products like vaccines and therapeutics. It works by taking advantage of the size difference between viruses and larger molecules like antibodies or proteins. Viruses are much smaller than most cells and cell debris; even the largest viruses are often less than 0.1 microns in size. Nanofiltration utilizes nanometer-sized pores in filter membranes which allowdesired product molecules to pass through while blocking viruses from passing based on their larger size.

How Does Virus Filtration Work?

In nanofiltration, the fluid containing the biological product is passed through a filter with a precise pore size. These Virus Filtration contain specially engineered membranes with tiny cylindrical pores ranging in size from 15-35 nanometers depending on the specific viruses targeted for removal. As the fluid passes through under pressure, desired larger molecules like antibodies freely flow through the pores. However, viruses, which are larger than the pore size, are unable to pass and are retained on the surface of the membrane filter. This effectively separates viruses from the therapeutic product. The filtered product stream is then collected for further processing or final formulation while the viruses are retained upstream of the filter.

Key Factors in Virus Filter Design and Performance

Several factors influence the design and performance of nanofiltration systems:

- Pore size - As mentioned, the pore size of the filter membrane must be smaller than the target viruses to effectively block their passage. Pore sizes typically range from 15-35nm.

- Flow rate - Higher flow rates increase throughput but can also increase the likelihood of viruses bypassing the filter if they deform or aggregate. Optimal flow rates balance productivity and clearance.

- Compressibility - Compressible membranes allow higher flow rates while maintaining exclusion. More compressible membranes are preferred.

- Virus characteristics - Size, shape, aggregation state and envelope vs. naked capsid all influence how effectively a virus is blocked. Filter optimization considers known product virus profiles.

- Product characteristics - Viscosity, particle content and other attributes affect flow properties and potential for fouling or virus aggregation. Filters are validated for specific product streams.

- Integrity testing - Post-filtration testing confirms membrane integrity was maintained and no defects allow virus breakthrough.

Validating Virus Clearance

To quantitatively demonstrate a Virus Filtration step provides adequate safety margins, validation uses a model virus spiked into product and measured before and after filtration. A log reduction value

(LRV) is determined from viral infectivity or DNA/RNA copy number reduction. For biologics, regulatory guidelines generally require ≥4-6 log reduction of a model virus that represents a known or potential contaminant. Ongoing process monitoring also confirms ongoing virus clearance capability is maintained.

Model Viruses for Validation

Common model viruses used to validate nanofiltration include:

- PhiX174 Bacteriophage - Small, non-enveloped DNA virus representative of parvoviruses.

- MS2 Bacteriophage - Similar size to noroviruses, a leading viral cause of food poisoning.

- PP7 Bacteriophage - Representative of small non-enveloped viruses like hepatitis A.

- Vesicular Stomatitis Virus (VSV) - Enveloped RNA virus similar in size to large viruses like HIV, CMV, HBV.

- Pseudorabies Virus (PrV) - Larger enveloped DNA virus can represent larger viruses.

Ideally the model virus chosen should be representative in both size and physico-chemical properties of known or potential product contaminants. Combining validation data using a range of model viruses strengthens the virus safety case.

Advantages of Virus Filtration

Nanofiltration offers some important advantages over other virus clearance methods:

- High clearance from early processing - As viruses are removed directly from the starting material or harvest, subsequent downstream steps have significantly reduced virus load to clear.

- Independent of product attributes - Performance is unaffected by characteristics of the product like pH, ionic strength or impurities compared to methods like pasteurization.

- No inactivation required - Live intact viruses are removed rather than inactivated so product quality attributes are not impacted.

- Continuous operation possible - Once validated, production-scale nanofiltration systems allow continuous or intermittent operation for improved productivity.

- Robust and reproducible - Well characterized process with predictable, high log reduction capability across many product classes when properly applied and maintained.

For these reasons, Virus Filtration has become a widely adopted standard technology for primary virus clearance in manufacturing of biotherapeutics. When validated and operated correctly, it provides a highly effective physical barrier toeven the smallest and most challenging viruses.

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