Recent Advances in Virus Removal Membranes For Antibody Purification

Viruses are microscopic parasitic particles that exist in the environment-including water, air, soil-and within living organisms. They are responsible for a wide range of infectious diseases and cause thousands of deaths worldwide each year. Due to their small size (20–200 nm), remarkable environmental stability, low infectious dose, and potential for long-distance transmission, viruses present a significant biosafety risk.

To minimize the risk of viral contamination in biopharmaceutical products, it is essential to establish robust, effective, and reliable virus clearance strategies throughout the manufacturing process.

Conventional virus inactivation and removal methods include low-pH incubation, solvent/detergent (S/D) treatment, irradiation, and membrane filtration. However, each technique has inherent limitations. Low-pH treatment may compromise the stability of recombinant proteins, while solvent/detergent treatment is effective only against enveloped viruses and provides limited protection against non-enveloped viruses. Likewise, ultraviolet (UV) and gamma (γ) irradiation may damage cells, proteins, or other biologically active substances.

 

Among current virus clearance technologies, membrane filtration has emerged as one of the most promising approaches. It effectively removes viruses across a broad size range while maintaining excellent tolerance to various physical and chemical processing conditions. Nanofiltration has become a standard virus removal step in plasma-derived product manufacturing, particularly for small viruses such as parvovirus B19. This technology has demonstrated high removal efficiency against numerous model viruses, including human immunodeficiency virus (HIV), bovine viral diarrhea virus (BVDV), pseudorabies virus (PRV), canine parvovirus (CPV), encephalomyocarditis virus (EMCV), and hepatitis A virus (HAV).

This review summarizes the current understanding of virus filtration mechanisms, recent advances in virus removal membrane materials, and the major challenges and future prospects of membrane-based virus filtration technologies in antibody purification.

 

 

 

1. Separation Mechanisms of Virus Removal Membranes

The performance of virus removal membranes is governed by the complex interplay among membrane pore size, surface properties, and electrostatic interactions with viral particles. By integrating size exclusion, electrostatic repulsion, and adsorption mechanisms, virus removal membranes effectively capture and retain viral contaminants while allowing target biomolecules to pass through.

1.1 Size Exclusion

Size exclusion is one of the most widely adopted mechanisms for virus removal due to its high selectivity and filtration efficiency (Figure 1).

By carefully controlling membrane pore size, viruses of different dimensions can be effectively separated, thereby ensuring high product purity. Furthermore, this mechanism does not require the addition of chemical reagents, eliminating the risk of chemical contamination or residual impurities in the final product.

These advantages make size exclusion an ideal virus removal strategy for biopharmaceutical manufacturing, particularly in the production of therapeutic proteins, monoclonal antibodies, vaccines, and other biological products.

Figure 1. Virus Removal by the Size Exclusion Mechanism

 

1.2 Electrostatic Interaction

Electrostatic interaction represents another important virus separation mechanism and has been widely utilized for viral capture and removal (Figure 2).

This approach exploits differences in the adsorption forces between membrane materials and virus particles. By tailoring membrane surface properties, viruses can be efficiently captured through electrostatic attraction.

Typical electrostatic filtration materials include nanofibrous membranes, porous materials, and functionalized nanomaterials. Owing to their large specific surface area and abundant functional groups, these materials provide numerous active binding sites that enhance interactions with viral particles and improve virus retention.

 

Studies have demonstrated that several operating conditions significantly influence electrostatic virus removal performance:

1. Low pH and low ionic strength maximize virus retention by strengthening electrostatic interactions between the membrane surface and viral particles.

2. Divalent cations, such as Mg²⁺ and Ca²⁺, can reduce virus removal efficiency by weakening electrostatic interactions.

3. During flow interruption, virus retention depends primarily on electrostatic attraction and hydrophobic adsorption between the membrane and bacteriophage PP7. Under these conditions, virus removal membranes with low protein affinity generally exhibit lower Log Reduction Value (LRV) performance.

Figure 2. Electrostatic Interaction Mechanism in Virus Filtration

With the rapid growth of the biopharmaceutical industry, virus filtration has become an increasingly critical step in the purification and manufacturing of biologics. However, conventional virus filtration membranes are often limited by insufficient permeate flux, inadequate long-term stability, and a high susceptibility to membrane fouling, all of which can compromise process performance and product yield.

To address these challenges, researchers have devoted significant efforts to developing next-generation membrane materials with enhanced filtration efficiency, improved fouling resistance, and superior operational stability.

Recent advances include:

1. Zwitterionic SPP-grafted polyethersulfone (PES) ultrafiltration membranes, which improve membrane hydrophilicity and reduce protein adsorption.

2. Microporous PES membranes grafted with zwitterionic polymer brushes, providing enhanced antifouling performance while maintaining high virus retention.

3. PAN/PET cellulose-based microfiltration membranes, offering excellent mechanical strength and filtration performance.

4. Positively charged polyvinyl alcohol (PVA) membranes fabricated through electrospinning and in situ photocrosslinking, which enhance virus capture through electrostatic interactions.

5. Layered nanochannel membranes constructed from self-assembled liquid crystal (LC) nanostructured materials, representing an emerging membrane platform with highly controllable pore architecture and promising virus removal capability.

The continuous development of these advanced membrane materials is expected to significantly improve virus filtration performance while overcoming the limitations of conventional filtration media.

 

3. Commercial Virus Filters

Commercial virus filters are generally available in two primary configurations:

* Hollow fiber membrane modules

* Flat-sheet membrane cassettes

These filters are typically manufactured from polymers such as:

* Hydrophilic polyvinylidene fluoride (PVDF)

* Hydrophilic polyethersulfone (PES)

* Regenerated cellulose (RC)

Unlike naturally hydrophilic regenerated cellulose, PVDF and PES are inherently hydrophobic materials. During membrane filtration, hydrophobic surfaces tend to adsorb proteins, which can lead to pore blockage, membrane fouling, reduced permeate flux, and ultimately lower product recovery.

The primary foulants encountered during virus filtration include protein aggregates, residual DNA, partially denatured proteins, and other process-related impurities. Fouling not only decreases purification efficiency but may also negatively affect final product quality. Consequently, incorporating an appropriate prefiltration step is an effective strategy for minimizing membrane fouling and extending filter service life.

Although many hollow fiber virus filters are technically capable of operating in Tangential Flow Filtration (TFF) mode, most commercial virus filtration processes are currently performed under Dead-End Filtration (DFF) conditions.

Murine minute virus (MVM) is widely recognized as one of the most challenging model viruses for validating virus removal performance. Consequently, the biopharmaceutical industry has broadly adopted 20 nm virus-retentive filters as the standard for virus clearance validation, replacing filters with larger pore sizes.

 

4. Conclusion

Despite substantial progress in membrane design and manufacturing technologies, achieving consistently high virus removal efficiency remains a significant challenge.

Although optimization of membrane pore structure and material properties has improved filtration performance, existing technologies still encounter limitations when removing diverse virus species, particularly small or structurally unique viruses. A single physical size-exclusion mechanism is insufficient to address all virus removal requirements. Therefore, integrating multiple separation mechanisms-including electrostatic adsorption, surface functionalization, and chemical modification-will be essential for enhancing overall virus clearance performance.

For antibody purification processes, current virus removal membranes continue to face challenges related to membrane throughput, fouling resistance, and virus retention efficiency. Future research should focus on innovative membrane materials, advanced surface modification technologies, and optimized process design to further improve membrane performance and meet the increasingly stringent quality requirements of modern biopharmaceutical manufacturing.

 

References

Luo, X., Ye, H., Li, H., *et al.* Recent Advances in the Application of Virus Removal Membranes for Antibody Purification Processes. *Shandong Chemical Industry*, 2024, 53(19): 153–157. DOI: 10.19319/j.cnki.issn.1008-021X.2024.19.048.